Methods for enhancing the degradation or conversion of cellulosic material

ABSTRACT

The present invention relates to methods for degrading or converting a cellulosic material and for producing a substance from a cellulosic material.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/722,529, filed Sep. 30, 2005, which application is incorporatedherein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under NREL SubcontractNo. ZCO-30017-02, Prime Contract DE-AC36-98GO10337 awarded by theDepartment of Energy. The government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for degrading or converting acellulosic material and for producing a substance from a cellulosicmaterial.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently bonded bybeta-1,4-linkages. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of cellulosic feedstocks into ethanol has the advantagesof the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

It would be advantageous in the art to improve the ability to convertcellulosic feedstocks.

U.S. Published Patent Application Serial No. 2003/0113734. discloses anisolated cellulase protein, identified as EGVII, and nucleic acids whichencode EGVII.

It is an object of the present invention to provide isolatedpolypeptides having cellulolytic enhancing activity and isolated nucleicacid sequences encoding the polypeptides to improve the conversion ofcellulosic feedstocks.

SUMMARY OF THE INVENTION

The present invention relates to methods for degrading or converting acellulosic material, comprising: treating the cellulosic material withan effective amount of one or more cellulolytic proteins in the presenceof an effective amount of a polypeptide having cellulolytic enhancingactivity, wherein the presence of the polypeptide having cellulolyticenhancing activity increases the degradation of cellulosic materialcompared to the absence of the polypeptide having cellulolytic enhancingactivity and wherein the polypeptide having cellulolytic enhancingactivity is selected from the group consisting of:

(a) a polypeptide comprising[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ], wherein x is anyamino acid, x(4,5) is any amino acid at 4 or 5 contiguous positions, andx(3) is any amino acid at 3 contiguous positions;

(b) a polypeptide comprising an amino acid sequence which has at least70% identity with the mature polypeptide of SEQ ID NO: 2;

(c) a polypeptide encoded by a polynucleotide which hybridizes under atleast medium stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequencecomprising the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) a complementary strand of (i) or (ii); and

(d) a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of the mature polypeptide of SEQ IDNO: 2.

The present invention also relates to methods for producing a substance,comprising:

(A) saccharifying a cellulosic material with an effective amount of oneor more cellulolytic proteins in the presence of an effective amount ofa polypeptide having cellulolytic enhancing activity, wherein thepresence of the polypeptide having cellulolytic enhancing activityincreases the degradation of cellulosic material compared to the absenceof the polypeptide having cellulolytic enhancing activity and whereinthe polypeptide having cellulolytic enhancing activity is selected fromthe group consisting of:

-   -   (i) a polypeptide comprising        [ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ], wherein x        is any amino acid, x(4,5) is any amino acid at 4 or 5 contiguous        positions, and x(3) is any amino acid at 3 contiguous positions;        and    -   (ii) a polypeptide comprising an amino acid sequence which has        at least 70% identity with the mature polypeptide of SEQ ID NO:        2;    -   (iii) a polypeptide encoded by a polynucleotide which hybridizes        under at least medium stringency conditions with (a) the mature        polypeptide coding sequence of SEQ ID NO: 1, (b) the genomic DNA        sequence comprising the mature polypeptide coding sequence of        SEQ ID NO: 1, or (c) a complementary strand of (i) or (ii); and    -   (iv) a variant comprising a conservative substitution, deletion,        and/or insertion of one or more amino acids of the mature        polypeptide of SEQ ID NO: 2;

(B) fermenting the saccharified cellulosic material of step (a) with oneor more fermenting microorganisms; and

(C) recovering the substance from the fermentation.

In a preferred aspect, the mature polypeptide is amino acids 20 to 249of SEQ ID NO: 2. In another preferred aspect, the mature polypeptidecoding sequence is nucleotides 77 to 766 of SEQ ID NO: 1.

The present invention further relates to detergent compositionscomprising such polypeptides having cellulolytic enhancing activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the cDNA sequence and the deduced amino acid sequence of aTrichoderma reesei RutC30 (ATCC 56765) GH61B polypeptide havingcellulolytic enhancing activity (SEQ ID NOs: 1 and 2, respectively).Predicted introns are italicized. The predicted signal peptide isunderlined.

FIG. 2 shows a restriction map of pTr3337.

FIG. 3 shows a restriction map of pTr61B.

FIG. 4 shows the hydrolysis of PCS (P020502CS, 100 MF autoclaved, 10g/L) by Trichoderma reesei broth expressing Aspergillus oryzaebeta-glucosidase (CLF) with or without supplementation by Trichodermareesei GH61B. Hydrolysis was carried out at 50° C., pH 5.0 for theindicated times. Celluclast Plus loading was 2.5 or 3.125 mg per gram ofPCS and the mixture contained 2.5 mg of CLF and 0.625 mg of GH61B pergram of PCS.

FIG. 5 the hydrolysis of PCS (P020502CS, 100 MF autoclaved, 10 g/L) byCelluclast Plus supplemented with GH61B protein. Aspergillus oryzaebroth not containing recombinant protein (Jal250) was added as acontrol. Incubation was for 115 hours at 50° C.

DEFINITIONS

Cellulolytic enhancing activity: The term “cellulolytic enhancingactivity” is defined herein as a biological activity which enhances thehydrolysis of a cellulosic material by proteins having cellulolyticactivity. For purposes of the present invention, cellulolytic enhancingactivity is determined by measuring the increase in reducing sugars fromthe hydrolysis of a cellulosic material by cellulolytic protein underthe following conditions: 5.0 mg of cellulolytic protein/g of cellulosein PCS for 5-7 day at 50° C. in the presence and absence of 0.01-2.5 mgof cellulolytic enhancing activity per g of cellulose in PCS compared toa control hydrolysis with equal total protein loading withoutcellulolytic enhancing activity (5.01-7.5 mg of cellulolytic protein/gof cellulose in PCS). In a preferred aspect, a mixture of Celluclast®1.5L (Novozymes A/S, Bagsvaerd, Denmark) in the presence of 3%Aspergillus oryzae beta-glucosidase (recombinantly produced inAspergillus oryzae according to WO 02/095014) or 3% Aspergillusfumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzaeaccording to Example 22 of WO 02/095014) of cellulase protein loading isused as the source of the cellulolytic activity.

The polypeptides having cellulolytic enhancing activity have at least20%, preferably at least 40%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 70%, more preferablyat least 80%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 100% of the cellulolyticenhancing activity of the polypeptide of the mature polypeptide of SEQID NO: 2.

Cellulolytic activity: The term “cellulolytic activity” is definedherein as a biological activity which hydrolyzes a cellulosic material.For purposes of the present invention, cellulolytic activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by a cellulolytic mixture under the following conditions: 1-10mg of cellulolytic protein/g of cellulose in PCS for 5-7 day at 50° C.compared to a control hydrolysis without addition of cellulolyticprotein. In a preferred aspect, a mixture of Celluclast® 1.5L (NovozymesA/S, Bagsvaerd, Denmark) in the presence of 3% Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) or 3% Aspergillus fumigatus beta glucosidase(recombinantly produced in Aspergillus oryzae according to Example 22 ofWO 02/095014) of cellulase protein loading as the source of thecellulolytic activity.

Pre-treated corn stover: The term “PCS” or “Pre-treated Corn Stover” isdefined herein as a cellulosic material derived from corn stover bytreatment with heat and dilute acid. For purposes of the presentinvention, PCS is made by the method described in Example 1, orvariations thereof in time, temperature and amount of acid.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” is defined herein as a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. Presently, Henrissat lists theGH61 Family as unclassified indicating that properties such asmechanism, catalytic nucleophile/base, catalytic proton donors, and 3-Dstructure are not known for polypeptides belonging to this family.

Cellulosic material: The cellulosic material can be any materialcontaining cellulose. Cellulose is generally found, for example, in thestems, leaves, hulls, husks, and cobs of plants or leaves, branches, andwood of trees. The cellulosic material can also be, but is not limitedto, herbaceous material, agricultural residues, forestry residues,municipal solid wastes, waste paper, and pulp and paper mill residues.It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix.

In a preferred aspect, the cellulosic material is corn stover. Inanother preferred aspect, the cellulosic material is corn fiber. Inanother preferred aspect, the cellulosic material is rice straw. Inanother preferred aspect, the cellulosic material is paper and pulpprocessing waste. In another preferred aspect, the cellulosic materialis woody or herbaceous plants. In another preferred aspect, thecellulosic material is bagasse.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art. For example,physical pretreatment techniques can include various types of milling,irradiation, steaming/steam explosion, and hydrothermolysis; chemicalpretreatment techniques can include dilute acid, alkaline, organicsolvent, ammonia, sulfur dioxide, carbon dioxide, and pH-controlledhydrothermolysis; and biological pretreatment techniques can involveapplying lignin-solubilizing microorganisms (see, for example, Hsu,T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, DC, 179-212; Ghosh, P., and Singh, A., 1993, Physicochemicaland biological treatments for enzymatic/microbial conversion oflignocellulosic biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J.D., 1994, Pretreating lignocellulosic biomass: a review, in EnzymaticConversion of Biomass for Fuels Production, Himmel, M. E., Baker, J. O.,and Overend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, DC, chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson, L., andHahn-Hagerdal, B., 1996, Fermentation of lignocellulosic hydrolysatesfor ethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander,L., and Eriksson, K.-E. L., 1990, Production of ethanol fromlignocellulosic materials: State of the art, Adv. Biochem.Eng./Biotechnol. 42: 63-95).

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively associated. It is, therefore,preferred that the substantially pure polypeptide is at least 92% pure,preferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 96% pure, morepreferably at least 97% pure, more preferably at least 98% pure, evenmore preferably at least 99%, most preferably at least 99.5% pure, andeven most preferably 100% pure by weight of the total polypeptidematerial present in the preparation.

The polypeptides having cellulolytic enhancing activity are preferablyin a substantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods orby classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having cellulolytic enhancing activity that is in its finalform following translation and any post-translational modifications,such as N-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having cellulolytic enhancing activity.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of EMBOSS with gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 matrix. The output ofNeedle labeled “longest identity” is used as the percent identity and iscalculated as follows:(Identical Residues×100)/(Length of Alignment−Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo nucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443453) asimplemented in the Needle program of EMBOSS with gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL matrix. The output ofNeedle labeled “longest identity” is used as the percent identity and iscalculated as follows:(Identical Residues×100)/(Length of Alignment−Number of Gaps inAlignment)

Homologous sequence: The term “homologous sequence” is defined herein assequences with an E value (or expectancy score) of less than 0.001 usingthe blastp (for protein databases) or tblastn (for nucleic aciddatabases) algorithms with the BLOSUM62 matrix, wordsize 3, gapexistence cost 11, gap extension cost 1, no low complexity filtration,and the mature GH61B protein sequence as query”. See Altschul et al.,1997, Nucleic Acids Res. 25: 3389-3402.

Polypeptide Fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of the mature polypeptide of SEQ ID NO: 2, or ahomologous sequence thereof, wherein the fragment has cellulolyticenhancing activity. Preferably, a fragment of the mature polypeptide ofSEQ ID NO: 2 contains at least 200 amino acid residues, more preferablyat least 210 amino acid residues, and most preferably at least 220 aminoacid residues.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of the mature polypeptide coding sequence of SEQ ID NO: 1, or ahomologous sequence thereof, wherein the subsequence encodes apolypeptide fragment having cellulolytic enhancing activity. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 1contains at least 600 nucleotides, more preferably at least 630nucleotides, and most preferably at least 660 nucleotides.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide which is at least 20% pure, preferablyat least 40% pure, more preferably at least 60% pure, even morepreferably at least 80% pure, most preferably at least 90% pure, andeven most preferably at least 95% pure, as determined by agaroseelectrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively associated. A substantially pure polynucleotide may, however,include naturally occurring 5′ and 3′ untranslated regions, such aspromoters and terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, preferably at least 92% pure, morepreferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 97% pure, evenmore preferably at least 98% pure, most preferably at least 99%, andeven most preferably at least 99.5% pure by weight. The polynucleotidesare preferably in a substantially pure form. In particular, it ispreferred that the polynucleotides disclosed herein are in “essentiallypure form”, i.e., that the polynucleotide preparation is essentiallyfree of other polynucleotide material with which it is nativelyassociated. Herein, the term “substantially pure polynucleotide” issynonymous with the terms “isolated polynucleotide” and “polynucleotidein isolated form.” The polynucleotides may be of genomic, cDNA, RNA,semisynthetic, synthetic origin, or any combinations thereof.

cDNA: The term “cDNA” is defined herein as a DNA molecule which can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that areusually present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA which is processed through aseries of steps before appearing as mature spliced mRNA. These stepsinclude the removal of intron sequences by a process called splicing.cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide. Each controlsequence may be native or foreign to the nucleotide sequence encodingthe polypeptide or native or foreign to each other. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG and TGA. The coding sequence may be aDNA, cDNA, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the mature polypeptide ofSEQ ID NO: 2 or a homologous sequence thereof; as well as geneticmanipulation of the DNA encoding such a polypeptide. The modificationcan be substitutions, deletions and/or insertions of one or more aminoacids as well as replacements of one or more amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having cellulolytic enhancing activity produced byan organism expressing a modified nucleotide sequence of the maturepolypeptide coding sequence of SEQ ID NO: 1 or a homologous sequencethereof. The modified nucleotide sequence is obtained through humanintervention by modification of the nucleotide sequence disclosed in SEQID NO: 1 or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for degrading or converting acellulosic material, comprising: treating the cellulosic material withan effective amount of a cellulolytic protein in the presence of aneffective amount of the polypeptide having cellulolytic enhancingactivity, wherein the presence of the polypeptide having cellulolyticenhancing activity increases the degradation of cellulosic materialcompared to the absence of the polypeptide having cellulolytic enhancingactivity and wherein the polypeptide having cellulolytic enhancingactivity is selected from the group consisting of:

(a) a polypeptide comprising[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ], wherein x is anyamino acid, x(4,5), is any amino acid at 4 or 5 contiguous positions,and x(3) is any amino acid at 3 contiguous positions;

(b) a polypeptide comprising an amino acid sequence which has at least70% identity with the mature polypeptide of SEQ ID NO: 2;

(c) a polypeptide encoded by a polynucleotide which hybridizes under atleast medium stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequencecomprising the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) a complementary strand of (i) or (ii); and

(d) a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of the mature polypeptide of SEQ IDNO: 2.

The present invention further comprises recovering the degraded orconverted cellulosic material. Soluble products of degradation orconversion of the cellulosic material can be separated from theinsoluble cellulosic material using technology well known in the artsuch as centrifugation, filtration, and gravity settling.

The present invention also relates to methods for producing a substance,comprising: (A) saccharifying a cellulosic material with an effectiveamount of one or more cellulolytic proteins in the presence of aneffective amount of the polypeptide having cellulolytic enhancingactivity, wherein the presence of the polypeptide having cellulolyticenhancing activity increases the degradation of cellulosic materialcompared to the absence of the polypeptide having cellulolytic enhancingactivity and wherein the polypeptide having cellulolytic enhancingactivity is selected from the group consisting of: (i) a polypeptidecomprising [ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ], whereinx is any amino acid, x(4,5) is any amino acid at 4 or 5 contiguouspositions, and x(3) is any amino acid at 3 contiguous positions; (ii) apolypeptide comprising an amino acid sequence which has at least 70%identity with the mature polypeptide of SEQ ID NO: 2; (iii) apolypeptide encoded by a polynucleotide which hybridizes under at leastmedium stringency conditions with (a) the mature polypeptide codingsequence of SEQ ID NO: 1, (b) the genomic DNA sequence comprising themature polypeptide coding sequence of SEQ ID NO: 1, or (c) acomplementary strand of (a) or (b); and (iv) a variant comprising aconservative substitution, deletion, and/or insertion of one or moreamino acids of the mature polypeptide of SEQ ID NO: 2; (B) fermentingthe saccharified cellulosic material of step (a) with one or morefermenting microorganisms; and (C) recovering the substance from thefermentation.

The polypeptides having cellulolytic enhancing activity and host cellsdescribed herein may be used in the production of monosaccharides,disaccharides, and polysaccharides as chemical or fermentationfeedstocks from biomass for the production of ethanol, plastics, otherproducts or intermediates. In particular, the polypeptides and hostcells may be used to increase the value of processing residues (drieddistillers grain, spent grains from brewing, sugarcane bagasse, etc.) bypartial or complete solubilization of cellulose or hemicellulose. Inboosting the processing by cellulolytic proteins of cellulosic materialto glucose, xylose, mannose, galactose, and arabinose, their polymers,or products derived from them as described below, the polypeptideshaving cellulolytic enhancing activity may be in the form of a crudefermentation broth with or without the cells or in the form of asemi-purified or purified enzyme preparation. The cellulolytic enhancingprotein may be a monocomponent preparation, e.g., a Family 61 protein, amulticomponent protein preparation, e.g., a number of Family 61proteins, or a combination of multicomponent and monocomponent proteinpreparations. The cellulolytic enhancing proteins may boost the activityof cellulolytic proteins, either in the acid, neutral, or alkalinepH-range. Alternatively, a host cell may be used as a source of such apolypeptide in a fermentation process with the biomass. The host cellmay also contain native or heterologous genes that encode cellulolyticprotein as well as other enzymes useful in the processing of biomass.

Biomass can include, but is not limited to, wood resources, municipalsolid waste, wastepaper, crops, and crop residues (see, for example,Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman,editor), pp. 105-118, Taylor & Francis, Washington D.C.; Wyman, 1994,Bioresource Technology 50: 3-16; Lynd, 1990, Applied Biochemistry andBiotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progress inBioconversion of Lignocellulosics, in Advances in BiochemicalEngineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp.23-40, Springer-Verlag, New York).

The predominant polysaccharide in the primary cell wall of biomass iscellulose, the second most abundant is hemi-cellulose, and the third ispectin. The secondary cell wall, produced after the cell has stoppedgrowing, also contains polysaccharides and is strengthened by polymericlignin covalently cross-linked to hemicellulose. Cellulose is ahomopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan,while hemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

Polypeptides Having Cellulolytic Enhancing Activity and PolynucleotidesThereof

In a first aspect, the isolated polypeptides having cellulolyticenhancing activity, comprise the following motif:[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ],wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5contiguous positions, and x(3) is any amino acid at 3 contiguouspositions. In the above motif, the accepted IUPAC single letter aminoacid abbreviation is employed.

In a second aspect, the isolated polypeptides having cellulolyticenhancing activity have an amino acid sequence which has a degree ofidentity to the mature polypeptide of SEQ ID NO: 2 (i.e., the maturepolypeptide) of at least 75%, preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, 97%, 98%, or 99%,which have cellulolytic enhancing activity (hereinafter “homologouspolypeptides”). In a preferred aspect, the homologous polypeptides havean amino acid sequence which differs by ten amino acids, preferably byfive amino acids, more preferably by four amino acids, even morepreferably by three amino acids, most preferably by two amino acids, andeven most preferably by one amino acid from the mature polypeptide ofSEQ ID NO: 2.

A polypeptide having cellulolytic enhancing activity of the presentinvention preferably comprises the amino acid sequence of SEQ ID NO: 2or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In a preferred aspect, a polypeptidecomprises the amino acid sequence of SEQ ID NO: 2. In another preferredaspect, a polypeptide comprises the mature polypeptide of SEQ ID NO: 2.In another preferred aspect, a polypeptide comprises amino acids 20 to249 of SEQ ID NO: 2, or an allelic variant thereof; or a fragmentthereof that has cellulolytic enhancing activity. In another preferredaspect, a polypeptide comprises amino acids 20 to 249 of SEQ ID NO: 2.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragmentthereof that has cellulolytic enhancing activity. In another preferredaspect, a polypeptide consists of the amino acid sequence of SEQ ID NO:2. In another preferred aspect, a polypeptide consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, a polypeptideconsists of amino acids 20 to 249 of SEQ ID NO: 2 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In another preferred aspect, a polypeptide consists of amino acids 20 to249 of SEQ ID NO: 2.

In a third aspect, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity which are encoded bypolynucleotides which hybridize under at least very low stringencyconditions, preferably at least low stringency conditions, morepreferably at least medium stringency conditions, more preferably atleast medium-high stringency conditions, even more preferably at leasthigh stringency conditions, and most preferably at least very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the genomic DNA sequence comprising the maturepolypeptide coding sequence of SEQ ID NO: 1, (iii) a subsequence of (i)or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (J.Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). A subsequenceof the mature polypeptide coding sequence of SEQ ID NO: 1 contains atleast 100 contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmentwhich has cellulolytic enhancing activity. In a preferred aspect, themature polypeptide coding sequence is nucleotides 77 to 766 of SEQ IDNO: 1.

The nucleotide sequence of SEQ ID NO: 1 or a subsequence thereof; aswell as the amino acid sequence of SEQ ID NO: 2 or a fragment thereof,may be used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having cellulolytic enhancing activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or cDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 14, preferably at least25, more preferably at least 35, and most preferably at least 70nucleotides in length. It is, however, preferred that the nucleic acidprobe is at least 100 nucleotides in length. For example, the nucleicacid probe may be at least 200 nucleotides, preferably at least 300nucleotides, more preferably at least 400 nucleotides, or mostpreferably at least 500 nucleotides in length. Even longer probes may beused, e.g., nucleic acid probes which are at least 600 nucleotides, atleast preferably at least 700 nucleotides, more preferably at least 800nucleotides, or most preferably at least 900 nucleotides in length. BothDNA and RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other organisms may,therefore, be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having cellulolyticenhancing activity. Genomic or other DNA from such other organisms maybe separated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1, or a subsequence thereof, the carriermaterial is preferably used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1,the genomic DNA sequence comprising the mature polypeptide codingsequence of SEQ ID NO: 1, its complementary strand, or a subsequencethereof, under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 73 to 1259 of SEQ ID NO: 1. In anotherpreferred aspect, the nucleic acid probe is a polynucleotide sequencewhich encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof.In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is the polynucleotidesequence contained in plasmid pTr3337 which is contained in Escherichiacoli NRRL B-30878, wherein the polynucleotide sequence thereof encodes apolypeptide having cellulolytic enhancing activity. In another preferredaspect, the nucleic acid probe is the mature polypeptide coding regioncontained in plasmid pTr3337 which is contained in Escherichia coli NRRLB-30878.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a fourth aspect, the polypeptides having cellulolytic enhancingactivity can be artificial variants comprising a conservativesubstitution, deletion, and/or insertion of one or more amino acids ofthe mature polypeptide of SEQ ID NO: 2 or a homologous sequence thereof;or the mature polypeptide thereof. Preferably, amino acid changes are ofa minor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of one to about 30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, change the pH optimum, and the like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,cellulolytic enhancing activity) to identify amino acid residues thatare critical to the activity of the molecule. See also, Hilton et al.,1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme orother biological interaction can also be determined by physical analysisof structure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides which arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2 is 10, preferably9, more preferably 8, more preferably 7, more preferably at most 6, morepreferably 5, more preferably 4, even more preferably 3, most preferably2, and even most preferably 1.

A polypeptide having cellulolytic enhancing activity of the presentinvention may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a nucleotide sequence is produced by the source or by a strain inwhich the nucleotide sequence from the source has been inserted. In apreferred aspect, the polypeptide obtained from a given source issecreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having cellulolytic enhancingactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingcellulolytic enhancing activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellulolytic enhancing activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having cellulolyticenhancing activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingcellulolytic enhancing activity.

The polypeptide having cellulolytic enhancing activity may also be afungal polypeptide, and more preferably a yeast polypeptide such as aCandida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia polypeptide having cellulolytic enhancing activity; or morepreferably a filamentous fungal polypeptide such as an Acremonium,Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide having cellulolytic enhancing activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having cellulolyticenhancing activity.

In another preferred aspect, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Coprinus cinereus, Diplodia gossyppina,Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola insolens,Humicola lanuginosa, Magnaporthe grisea, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Pseudoplectania nigrella, Thermoascus aurantiacus,Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii,Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, orTrichophaea saccata polypeptide having cellulolytic enhancing activity.

In a more preferred aspect, the polypeptide is a Trichoderma reeseipolypeptide. In a most preferred aspect, the polypeptide is aTrichoderma reesei RutC30 (ATCC 56765) polypeptide, e.g., thepolypeptide with the amino acid sequence of SEQ ID NO: 2 or fragmentsthereof, e.g., the mature polypeptide.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides having cellulolytic enhancing activity also include fusedpolypeptides or cleavable fusion polypeptides in which anotherpolypeptide is fused at the N-terminus or the C-terminus of thepolypeptide or fragment thereof having cellulolytic enhancing activity.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) encoding a polypeptide h having cellulolyticenhancing activity. Techniques for producing fusion polypeptides areknown in the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.

Polynucleotides having nucleotide sequences which encode polypeptideshaving cellulolytic enhancing activity can be isolated and utilized topractice the methods of the present invention, as described herein.

In a preferred aspect, the nucleotide sequence is set forth in SEQ IDNO: 1. In another more preferred aspect, the nucleotide sequence is thesequence contained in plasmid pTr3337 which is contained in E. coli NRRLB-30878. In another preferred aspect, the nucleotide sequence is themature polypeptide coding region of SEQ ID NO: 1. In another preferredaspect, the nucleotide sequence is nucleotides 77 to 766 of SEQ IDNO: 1. In another more preferred aspect, the nucleotide sequence is themature polypeptide coding region contained in plasmid pTr3337 which iscontained in E. coli NRRL B-30878. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 2 or the mature polypeptide thereof,which differ from SEQ ID NO: 1 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 1 which encodefragments of SEQ ID NO: 2 that have cellulolytic enhancing activity.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 1, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 2. In a preferred aspect, the maturepolypeptide is amino acids 20 to 249 of SEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides from such genomic DNA can be effected, e.g., by usingthe well known polymerase chain reaction (PCR) or antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features. See, e.g., Innis et al., 1990, PCR: A Guide toMethods and Application, Academic Press, New York. Other nucleic acidamplification procedures such as ligase chain reaction (LCR), ligatedactivated transcription (LAT) and nucleotide sequence-basedamplification (NASBA) may be used. The polynucleotides may be clonedfrom a strain of Trichoderma, or another or related organism and thus,for example, may be an allelic or species variant of the polypeptideencoding region of the nucleotide sequence.

In the methods of the present invention, the polynucleotides havenucleotide sequences which have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 (i.e., nucleotides 388 to1332) of at least 75%, preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, 97%, 98%, or 99% identity, whichencode an active polypeptide.

Modification of a nucleotide sequence encoding a polypeptide havingcellulolytic enhancing activity may be necessary for the synthesis ofpolypeptides substantially similar to the polypeptide. The term“substantially similar” to the polypeptide refers to non-naturallyoccurring forms of the polypeptide. These polypeptides may differ insome engineered way from the polypeptide isolated from its nativesource, e.g., artificial variants that differ in specific activity,thermostability, pH optimum, or the like. The variant sequence may beconstructed on the basis of the nucleotide sequence presented as thepolypeptide encoding region of SEQ ID NO: 1, e.g., a subsequencethereof, and/or by introduction of nucleotide substitutions which do notgive rise to another amino acid sequence of the polypeptide encoded bythe nucleotide sequence, but which correspond to the codon usage of thehost organism intended for production of the enzyme, or by introductionof nucleotide substitutions which may give rise to a different aminoacid sequence. For a general description of nucleotide substitution,see, e.g., Ford et al., 1991, Protein Expression and Purification 2:95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, Science 244: 1081-1085). In the lattertechnique, mutations are introduced at every positively charged residuein the molecule, and the resultant mutant molecules are tested forcellulolytic enhancing activity to identify amino acid residues that arecritical to the activity of the molecule. Sites of substrate-enzymeinteraction can also be determined by analysis of the three-dimensionalstructure as determined by such techniques as nuclear magnetic resonanceanalysis, crystallography or photoaffinity labeling (see, e.g., de Voset al., 1992, Science 255: 306-312; Smith et al., 1992, Journal ofMolecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309:59-64).

The polynucleotide may be a polynucleotide encoding a polypeptide havingcellulolytic enhancing activity that hybridize under at least very lowstringency conditions, preferably at least low stringency conditions,more preferably at least medium stringency conditions, more preferablyat least medium-high stringency conditions, even more preferably atleast high stringency conditions, and most preferably at least very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the genomic DNA sequence comprising the maturepolypeptide coding sequence of SEQ ID NO: 1, or (iii) a complementarystrand of (i) or (ii); or allelic variants and subsequences thereof(Sambrook et al., 1989, supra), as defined herein. In a preferredaspect, the mature polypeptide coding sequence is nucleotides 77 to 766of SEQ ID NO: 1.

Nucleic Acid Constructs

An isolated polynucleotide encoding a polypeptide having cellulolyticenhancing activity may be manipulated in a variety of ways to providefor expression of the polypeptide by constructing a nucleic acidconstruct comprising an isolated polynucleotide encoding a polypeptidehaving cellulolytic enhancing activity operably linked to one or morecontrol sequences which direct the expression of the coding sequence ina suitable host cell under conditions compatible with the controlsequences. Manipulation of the polynucleotide's sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying polynucleotide sequencesutilizing recombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide having cellulolytic enhancingactivity. The promoter sequence contains transcriptional controlsequences which mediate the expression of the polypeptide. The promotermay be any nucleotide sequence which shows transcriptional activity inthe host cell of choice including mutant, truncated, and hybridpromoters, and may be obtained from genes encoding extracellular orintracellular polypeptides either homologous or heterologous to the hostcell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs, especially in a bacterial host cell, are thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in a filamentous fungal host cell are promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice, i.e.,secreted into a culture medium, may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

In a preferred aspect, the signal peptide comprises or consists of aminoacids 1 to 19 of SEQ ID NO: 2. In another preferred aspect, the signalpeptide coding region comprises or consists of nucleotides 20 to 76 ofSEQ ID NO: 1.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The various nucleic acids and control sequences described herein may bejoined together to produce a recombinant expression vector comprising apolynucleotide encoding a polypeptide having cellulolytic enhancingactivity, a promoter, and transcriptional and translational stopsignals. The expression vectors may include one or more convenientrestriction sites to allow for insertion or substitution of thenucleotide sequence encoding the polypeptide at such sites.Alternatively, a polynucleotide encoding a polypeptide havingcellulolytic enhancing activity may be expressed by inserting thenucleotide sequence or a nucleic acid construct comprising the sequenceinto an appropriate vector for expression. In creating the expressionvector, the coding sequence is located in the vector so that the codingsequence is operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors preferably contain one or more selectable markers whichpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors preferably contain an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAM91 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide encoding a polypeptide havingcellulolytic enhancing activity may be inserted into the host cell toincrease production of the polypeptide. An increase in the copy numberof the polynucleotide can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the polynucleotidewhere cells containing amplified copies of the selectable marker gene,and thereby additional copies of the polynucleotide, can be selected forby cultivating the cells in the presence of the appropriate selectableagent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

Host Cells

Recombinant host cells, comprising a polynucleotide encoding apolypeptide having cellulolytic enhancing activity, can beadvantageously used in the recombinant production of the polypeptide. Avector comprising such a polynucleotide is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

The bacterial host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyceslividans.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98:6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarum sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide having cellulolytic enhancing activity, comprising (a)cultivating a cell, which in its wild-type form is capable of producingthe polypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide. Preferably, the cell isof the genus Trichoderma, more preferably Trichoderma reesei, and mostpreferably Trichoderma reesei RutC30.

The present invention also relates to methods for producing apolypeptide having cellulolytic enhancing activity, comprising (a)cultivating a recombinant host cell under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide having cellulolytic enhancing activity, comprising (a)cultivating a host cell under conditions conducive for production of thepolypeptide, wherein the host cell comprises a mutant nucleotidesequence having at least one mutation in the mature polypeptide codingregion of SEQ ID NO: 1, wherein the mutant nucleotide sequence encodes apolypeptide which consists of the mature polypeptide of SEQ ID NO: 2,and (b) recovering the polypeptide. In a preferred aspect, the maturepolypeptide of SEQ ID NO: 2 is amino acids 20 to 249 of SEQ ID NO: 2.

In the production methods, the cells are cultivated in a nutrient mediumsuitable for production of the polypeptide using methods well known inthe art. For example, the cell may be cultivated by shake flaskcultivation, and small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the polypeptide to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides having cellulolytic enhancing activity are detectedusing the methods described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

Cellulolytic Proteins

In the methods of the present invention, the cellulolytic protein may beany protein involved in the processing of cellulosic material toglucose, or hemicellulose to xylose, mannose, galactose, and arabinose,their polymers, or products derived from them as described below. Thecellulolytic protein may be a monocomponent preparation, e.g., acellulase, a multicomponent preparation, e.g., endoglucanase,cellobiohydrolase, glucohydrolase, beta-glucosidase, as defined below,or a combination of multicomponent and monocomponent proteinpreparations. The cellulolytic proteins may have activity, i.e.,hydrolyze cellulose, either in the acid, neutral, or alkaline pH-range.

The cellulolytic protein may be of fungal or bacterial origin, which maybe obtainable or isolated and purified from microorganisms which areknown to be capable of producing cellulolytic enzymes, e.g., species ofBacillus, Pseudomonas, Humicola, Coprinus, Thielavia, Fusarium,Myceliophthora, Acremonium, Cephalosporium, Scytalidium, Penicillium orAspergillus (see, for example, EP 458162), especially those produced bya strain selected from Humicola insolens (reclassified as Scytalidiumthermophilum, see for example, U.S. Pat. No. 4,435,307), Coprinuscinereus, Fusarium oxysporum, Myceliophthora thermophila, Meripilusgiganteus, Thielavia terrestris, Acremonium sp., Acremonium persicinum,Acremonium acremonium, Acremonium brachypenium, Acremoniumdichromosporum, Acremonium obclavatum, Acremonium pinkertoniae,Acremonium roseogriseum, Acremonium incoloratum, and Acremonium furatum;preferably from Humicola insolens DSM 1800, Fusarium oxysporum DSM 2672,Myceliophthora thermophila CBS 117.65, Cephalosporium sp. RYM-202,Acremonium sp. CBS 478.94, Acremonium sp. CBS 265.95, Acremoniumpersicinum CBS 169.65, Acremonium acremonium AHU 9519, Cephalosporiumsp. CBS 535.71, Acremonium brachypenium CBS 866.73, Acremoniumdichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74, Acremoniumpinkertoniae CBS 157.70, Acremonium roseogriseum CBS 134.56, Acremoniumincoloratum CBS 146.62, and Acremonium furatum CBS 299.70H. Cellulolyticproteins may also be obtained from Trichoderma (particularly Trichodermaviride, Trichoderma reesei, and Trichoderma koningii), alkalophilicBacillus (see, for example, U.S. Pat. No. 3,844,890 and EP 458162), andStreptomyces (see, for example, EP 458162). Chemically modified orprotein engineered mutants of cellulolytic proteins may also be used.

Especially suitable cellulolytic proteins are the alkaline or neutralcellulases. Examples of such cellulases are cellulases described in EP495,257, EP 531,372, WO 96/11262, WO 96/29397, WO 98/08940. Otherexamples are cellulase variants such as those described in WO 94/07998,EP 531,315, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,763,254, U.S. Pat. No. 5,776,757, WO 89/09259, WO 95/24471,WO 98/12307, and

The cellulolytic proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, CA, 1991). Suitable media are available from commercial suppliersor may be prepared according to published compositions (e.g., incatalogues of the American Type Culture Collection). Temperature rangesand other conditions suitable for growth and cellulolytic proteinproduction are known in the art (see, e.g., Bailey, J. E., and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY,1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of a cellulolytic protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe cellulolytic protein or cellulolytic enhancing protein to beexpressed or isolated.

The resulting cellulolytic proteins produced by the methods describedabove may be recovered from the fermentation medium and purified byconventional procedures as described herein.

Cellulolytic protein may hydrolyze or hydrolyzes carboxymethyl cellulose(CMC), thereby decreasing the viscosity of the incubation mixture. Theresulting reduction in viscosity may be determined by a vibrationviscosimeter (e.g., MIVI 3000 from Sofraser, France). Determination ofcellulase activity, measured in terms of Cellulase Viscosity Unit(CEVU), quantifies the amount of catalytic activity present in a sampleby measuring the ability of the sample to reduce the viscosity of asolution of carboxymethyl cellulose (CMC). The assay is performed at thetemperature and pH suitable for the cellulolytic protein and substrate.For Celluclast™ (Novozymes A/S, Bagsvaerd, Denmark) the assay is carriedout at 40° C. in 0.1 M phosphate pH 9.0 buffer for 30 minutes with CMCas substrate (33.3 g/L carboxymethyl cellulose Hercules 7 LFD) and anenzyme concentration of approximately 3.3-4.2 CEVU/ml. The CEVU activityis calculated relative to a declared enzyme standard, such as CELLUZYME™Standard 17-1194 (obtained from Novozymes A/S, Bagsvaerd, Denmark).

Examples of cellulolytic preparations suitable for use in the presentinvention include, for example, CELLUCLAST™ (available from NovozymesA/S) and NOVOZYM™ 188 (available from Novozymes A/S). Other commerciallyavailable preparations comprising cellulase which may be used includeCELLUZYME™, CEREFLO™ and ULTRAFLO™ (Novozymes A/S), LAMINEX™ andSPEZYME™ CP (Genencor Int.), and ROHAMENT™ 7069 W (Rohm GmbH). Thecellulase enzymes are added in amounts effective from about 0.001% toabout 5.0% wt. of solids, more preferably from about 0.025% to about4.0% wt. of solids, and most preferably from about 0.005% to about 2.0%wt. of solids.

As mentioned above, the cellulolytic proteins or cellulolytic enhancingproteins used in the methods of the present invention may bemonocomponent preparations, i.e., a component essentially free of othercellulolytic components. The single component may be a recombinantcomponent, i.e., produced by cloning of a DNA sequence encoding thesingle component and subsequent cell transformed with the DNA sequenceand expressed in a host (see, for example, WO 91/17243 and WO 91/17244).Other examples of monocomponent cellulolytic proteins include, but arenot limited to, those disclosed in JP-07203960-A and WO-9206209. Thehost is preferably a heterologous host (enzyme is foreign to host), butthe host may under certain conditions also be a homologous host (enzymeis native to host). Monocomponent cellulolytic proteins may also beprepared by purifying such a protein from a fermentation broth.

Examples of monocomponent cellulolytic proteins useful in practicing themethods of the present invention include, but are not limited to,endoglucanase, cellobiohydrolase, glucohydrolase, and beta-glucosidase.

The term “endoglucanase” is defined herein as anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. No. 3.2.1.4),which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

The exo-1,4-beta-D-glucanases include both cellobiohydrolases andglucohydrolases. For purposes of the present invention, exoglucanaseactivity is determined according to the procedure described by Himmel etal., 1986, J. Biol. Chem. 261: 12948-12955.

The term “cellobiohydrolase” is defined herein as a 1,4-beta-D-glucancellobiohydrolase (E.C. 3.2.1.91), which catalyzes the hydrolysis of1,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, orany beta-1,4-linked glucose containing polymer, releasing cellobiosefrom the reducing or non-reducing ends of the chain. For purposes of thepresent invention, cellobiohydrolase activity is determined according tothe procedures described by Lever et al., 1972, Anal. Biochem. 47:273-279 and by van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156;van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288. In thepresent invention, the Lever et al. method was employed to assesshydrolysis of cellulose in corn stover, while the method of vanTilbeurgh et al. was used to determine the cellobiohydrolase activity ona fluorescent disaccharide derivative.

The term “glucohydrolase” is defined herein as a 1,4-beta-D-glucanglucohydrolase (E.C. 3.2.1.74), which catalyzes the hydrolysis of1,4-linkages (O-glycosyl bonds) in 1,4-beta-D-glucans so as to removesuccessive glucose units.

The term “beta-glucosidase” is defined herein as a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis ofterminal non-reducing beta-D-glucose residues with the release ofbeta-D-glucose. For purposes of the present invention, beta-glucosidaseactivity is determined according to the basic procedure described byVenturi et al., 2002, J. Basic Microbiol. 42: 55-66, except differentconditions were employed as described herein. One unit ofbeta-glucosidase activity is defined as 1.0 μmole of p-nitrophenolproduced per minute at 50° C., pH 5 from 4 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate, 0.01% Tween-20.

Processing of Cellulosic Material

The methods of the present invention can be used to process a cellulosicmaterial to many useful substances, e.g., chemicals and fuels. Inaddition to ethanol, some commodity and specialty chemicals that can beproduced from cellulose include xylose, acetone, acetate, glycine,lysine, organic acids (e.g., lactic acid), 1,3-propanediol, butanediol,glycerol, ethylene glycol, furfural, polyhydroxyalkanoates, andcis,cis-muconic acid (Lynd, L. R., Wyman, C. E., and Gerngross, T. U.,1999, Biocommodity Engineering, Biotechnol. Prog., 15: 777-793;Philippidis, G. P., 1996, Cellulose bioconversion technology, inHandbook on Bioethanol: Production and Utilization, Wyman, C. E., ed.,Taylor & Francis, Washington, DC, 179-212; and Ryu, D. D. Y., andMandels, M., 1980, Cellulases: biosynthesis and applications, Enz.Microb. Technol., 2: 91-102). Potential coproduction benefits extendbeyond the synthesis of multiple organic products from fermentablecarbohydrate. Lignin-rich residues remaining after biological processingcan be converted to lignin-derived chemicals, or used for powerproduction.

Conventional methods used to process the cellulosic material inaccordance with the methods of the present invention are well understoodto those skilled in the art. The methods of the present invention may beimplemented using any conventional biomass processing apparatusconfigured to operate in accordance with the invention.

Such an apparatus may include a batch-stirred reactor, a continuous flowstirred reactor with ultrafiltration, a continuous plug-flow columnreactor (Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of theenzymatic hydrolysis of cellulose: 1. A mathematical model for a batchreactor process, Enz. Microb. Technol. 7: 346-352), an attrition reactor(Ryu, S. K., and Lee, J. M., 1983, Bioconversion of waste cellulose byusing an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65), or areactor with intensive stirring induced by an electromagnetic field(Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y.,Protas, O. V., 1996, Enhancement of enzymatic cellulose hydrolysis usinga novel type of bioreactor with intensive stirring induced byelectromagnetic field, Appl. Biochem. Biotechnol. 56: 141-153).

The conventional methods include, but are not limited to,saccharification, fermentation, separate hydrolysis and fermentation(SHF), simultaneous saccharification and fermentation (SSF),simultaneous saccharification and cofermentation (SSCF), hybridhydrolysis and fermentation (HHF), and direct microbial conversion(DMC).

SHF uses separate process steps to first enzymatically hydrolyzecellulose to glucose and then ferment glucose to ethanol. In SSF, theenzymatic hydrolysis of cellulose and the fermentation of glucose toethanol is combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC,179-212). SSCF includes the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF includes two separate steps carried out in the same reactor but atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(cellulase production, cellulose hydrolysis, and fermentation) in onestep (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S.,2002, Microbial cellulose utilization: Fundamentals and biotechnology,Microbiol. Mol. Biol. Reviews 66: 506-577).

“Fermentation” or “fermentation process” refers to any fermentationprocess or any process comprising a fermentation step. A fermentationprocess includes, without limitation, fermentation processes used toproduce fermentation products including alcohols (e.g., arabinitol,butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol, andxylitol); organic acids (e.g., acetic acid, acetonic acid, adipic acid,ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid,fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaricacid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,malonic acid, oxalic acid, propionic acid, succinic acid, and xylonicacid); ketones (e.g., acetone); amino acids (e.g., aspartic acid,glutamic acid, glycine, lysine, serine, and threonine); gases (e.g.,methane, hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)).Fermentation processes also include fermentation processes used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry, and tobacco industry.

The polypeptide having cellulolytic enhancing activity may be in theform of a crude fermentation broth with or without the cells or in theform of a semi-purified or purified enzyme preparation. The cellulolyticenhancing protein may be a monocomponent preparation, e.g., a Family 61protein, a multicomponent protein preparation, e.g., a number of Family61 proteins, or a combination of multicomponent and monocomponentprotein preparations.

The substance can be any substance derived from the fermentation. In apreferred aspect, the substance is an alcohol. It will be understoodthat the term “alcohol” encompasses a substance that contains one ormore hydroxyl moieties. In a more preferred aspect, the alcohol isarabinitol. In another more preferred aspect, the alcohol is butanol. Inanother more preferred aspect, the alcohol is ethanol. In another morepreferred aspect, the alcohol is glycerol. In another more preferredaspect, the alcohol is methanol. In another more preferred aspect, thealcohol is 1,3-propanediol. In another more preferred aspect, thealcohol is sorbitol. In another more preferred aspect, the alcohol isxylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R.,2002, The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the substance is an organic acid. Inanother more preferred aspect, the organic acid is acetic acid. Inanother more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid: Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the substance is a ketone. It will beunderstood that the term “ketone” encompasses a substance that containsone or more ketone moieties. In another more preferred aspect, theketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.

In another preferred aspect, the substance is an amino acid. In anothermore preferred aspect, the organic acid is aspartic acid. In anothermore preferred aspect, the amino acid is glutamic acid. In another morepreferred aspect, the amino acid is glycine. In another more preferredaspect, the amino acid is lysine. In another more preferred aspect, theamino acid is serine. In another more preferred aspect, the amino acidis threonine. See, for example, Richard, A., and Margaritis, A., 2004,Empirical modeling of batch fermentation kinetics for poly(glutamicacid) production and other microbial biopolymers, Biotechnology andBioengineering 87 (4): 501-515.

In another preferred aspect, the substance is a gas. In another morepreferred aspect, the gas is methane. In another more preferred aspect,the gas is H₂. In another more preferred aspect, the gas is CO₂. Inanother more preferred aspect, the gas is CO. See, for example, Kataoka,N., A. Miya, and K. Kiriyama, 1997, Studies on hydrogen production bycontinuous culture system of hydrogen-producing anaerobic bacteria,Water Science and Technology 36 (6-7): 4147; and Gunaseelan V. N. inBiomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobicdigestion of biomass for methane production: A review.

Production of a substance from cellulosic material typically requiresfour major steps. These four steps are pretreatment, enzymatichydrolysis, fermentation, and recovery. Exemplified below is a processfor producing ethanol, but it will be understood that similar processescan be used to produce other substances, for example, the substancesdescribed above.

Pretreatment. In the pretreatment or pre-hydrolysis step, the cellulosicmaterial is heated to break down the lignin and carbohydrate structure,solubilize most of the hemicellulose, and make the cellulose fractionaccessible to cellulolytic enzymes. The heating is performed eitherdirectly with steam or in slurry where a catalyst may also be added tothe material to speed up the reactions. Catalysts include strong acids,such as sulfuric acid and SO₂, or alkali, such as sodium hydroxide. Thepurpose of the pre-treatment stage is to facilitate the penetration ofthe enzymes and microorganisms. Cellulosic biomass may also be subjectto a hydrothermal steam explosion pre-treatment (See U.S. PatentApplication No. 20020164730).

Saccharification. In the enzymatic hydrolysis step, also known assaccharification, enzymes as described herein are added to thepretreated material to convert the cellulose fraction to glucose and/orother sugars. The saccharification is generally performed instirred-tank reactors or fermentors under controlled pH, temperature,and mixing conditions. A saccharification step may last up to 200 hours.Saccharification may be carried out at temperatures from about 30° C. toabout 65° C., in particular around 50° C., and at a pH in the rangebetween about 4 and about 5, especially around pH 4.5. To produceglucose that can be metabolized by yeast, the hydrolysis is typicallyperformed in the presence of a beta-glucosidase.

Fermentation. In the fermentation step, sugars, released from thecellulosic material as a result of the pretreatment and enzymatichydrolysis steps, are fermented to ethanol by a fermenting organism,such as yeast. The fermentation can also be carried out simultaneouslywith the enzymatic hydrolysis in the same vessel, again under controlledpH, temperature, and mixing conditions. When saccharification andfermentation are performed simultaneously in the same vessel, theprocess is generally termed simultaneous saccharification andfermentation or SSF.

Any suitable cellulosic substrate or raw material may be used in afermentation process in practicing the present invention. The substrateis generally selected based on the desired fermentation product, i.e.,the substance to be obtained from the fermentation, and the processemployed, as is well known in the art. Examples of substrates suitablefor use in the methods of present invention, includecellulose-containing materials, such as wood or plant residues or lowmolecular sugars DP1-3 obtained from processed cellulosic material thatcan be metabolized by the fermenting microorganism, and which may besupplied by direct addition to the fermentation medium.

The term “fermentation medium” will be understood to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism suitable for usein a desired fermentation process. Suitable fermenting microorganismsaccording to the invention are able to ferment, i.e., convert, sugars,such as glucose, xylose, arabinose, mannose, galactose, oroligosaccharides directly or indirectly into the desired fermentationproduct. Examples of fermenting microorganisms include fungal organisms,such as yeast. Preferred yeast includes strains of the Saccharomycesspp., and in particular, Saccharomyces cerevisiae. Commerciallyavailable yeast include, e.g., Red Star®/™/Lesaffre Ethanol Red(available from Red Star/Lesaffre, USA) FALI (available fromFleischmann's Yeast, a division of Burns Philp Food Inc., USA),SUPERSTART (available from Alltech), GERT STRAND (available from GertStrand AB, Sweden) and FERMIOL (available from DSM Specialties).

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida pseudotropicalis. In another more preferredaspect, the yeast is Candida brassicae. In another preferred aspect, theyeast is a Clavispora. In another more preferred aspect, the yeast isClavispora lusitaniae. In another more preferred aspect, the yeast isClavispora opuntiae. In another preferred aspect, the yeast is aPachysolen. In another more preferred aspect, the yeast is Pachysolentannophilus. In another preferred aspect, the yeast is a Bretannomyces.In another more preferred aspect, the yeast is Bretannomyces clausenii(Philippidis, G. P., 1996, Cellulose bioconversion technology, inHandbook on Bioethanol: Production and Utilization, Wyman, C. E., ed.,Taylor & Francis, Washington, DC, 179-212).

Bacteria that can efficiently ferment glucose to ethanol include, forexample, Zymomonas mobilis and Clostridium thermocellum (Philippidis,1996, supra).

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The cloning of heterologous genes in Saccharomyces cerevisiae (Chen, Z.,Ho, N. W. Y., 1993, Cloning and improving the expression of Pichiastipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.Biochem. Biotechnol. 3940: 135-147; Ho, N. W. Y., Chen, Z, Brainard, A.P., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859), or in bacteria such as Escherichia coli (Beall, D. S.,Ohta, K., Ingram, L. O., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303), Klebsiella oxytoca (Ingram, L. O., Gomes, P. F.,Lai, X., Moniruzzaman, M., Wood, B. E., Yomano, L. P., York, S. W.,1998, Metabolic engineering of bacteria for ethanol production,Biotechnol. Bioeng. 58: 204-214), and Zymomonas mobilis (Zhang, M.,Eddy, C., Deanda, K., Finkelstein, M., and Picataggio, S., 1995,Metabolic engineering of a pentose metabolism pathway in ethanologenicZymomonas mobilis, Science 267: 240-243; Deanda, K., Zhang, M., Eddy,C., and Picataggio, S., 1996, Development of an arabinose-fermentingZymomonas mobilis strain by metabolic pathway engineering, Appl.Environ. Microbiol. 62: 4465-4470) has led to the construction oforganisms capable of converting hexoses and pentoses to ethanol(cofermentation).

Yeast or another microorganism typically is added to the degradedcellulose or hydrolysate and the fermentation is performed for about 24to about 96 hours, such as about 35 to about 60 hours. The temperatureis typically between about 26° C. to about 40° C., in particular atabout 32° C., and at about pH 3 to about pH 6, in particular around pH4-5.

In a preferred aspect, yeast or another microorganism is applied to thedegraded cellulose or hydrolysate and the fermentation is performed forabout 24 to about 96 hours, such as typically 35-60 hours. In apreferred aspect, the temperature is generally between about 26 to about40° C., in particular about 32° C., and the pH is generally from aboutpH 3 to about pH 6, preferably around pH 4-5. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 5×10⁷ viable count per ml of fermentation broth. During anethanol producing phase the yeast cell count should preferably be in therange from approximately 10⁷ to 10¹⁰, especially around approximately2×10⁸. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

The most widely used process in the art is the simultaneoussaccharification and fermentation (SSF) process where there is noholding stage for the saccharification, meaning that yeast and enzymeare added together.

For ethanol production, following the fermentation the mash is distilledto extract the ethanol. The ethanol obtained according to the process ofthe invention may be used as, e.g., fuel ethanol, drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator may be used in combination with any of theenzymatic processes described herein to further improve the fermentationprocess, and in particular, the performance of the fermentingmicroorganism, such as, rate enhancement and ethanol yield. A“fermentation stimulator” refers to stimulators for growth of thefermenting microorganisms, in particular, yeast. Preferred fermentationstimulators for growth include vitamins and minerals. Examples ofvitamins include multivitamins, biotin, pantothenate, nicotinic acid,meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid,riboflavin, and Vitamins A, B, C, D, and E. See, e.g., Alfenore et al.,Improving ethanol production and viability of Saccharomyces cerevisiaeby a vitamin feeding strategy during fed-batch process, Springer-Verlag(2002), which is hereby incorporated by reference. Examples of mineralsinclude minerals and mineral salts that can supply nutrients comprisingP, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Recovery. The alcohol is separated from the fermented cellulosicmaterial and purified by conventional methods of distillation. Ethanolwith a purity of up to about 96 vol. % can be obtained, which can beused as, for example, fuel ethanol, drinking ethanol, i.e., potableneutral spirits, or industrial ethanol.

For other substances, any method known in the art can be used including,but not limited to, chromatography (e.g., ion exchange, affinity,hydrophobic, chromatofocusing, and size exclusion), electrophoreticprocedures (e.g., preparative isoelectric focusing), differentialsolubility (e.g., ammonium sulfate precipitation), SDS-PAGE,distillation, or extraction.

In the methods of the present invention, the cellulolytic protein(s) andcellulolytic enhancing polypeptide(s) may be supplemented by one or moreadditional enzyme activities to improve the degradation of thecellulosic material. Preferred additional enzymes are hemicellulases,esterases (e.g., lipases, phospholipases, and/or cutinases), proteases,laccases, peroxidases, or mixtures thereof.

In the methods of the present invention, the additional enzyme(s) may beadded prior to or during fermentation, including during or after thepropagation of the fermenting microorganism(s).

The enzymes referenced herein may be derived or obtained from anysuitable origin, including, bacterial, fungal, yeast or mammalianorigin. The term “obtained” means herein that the enzyme may have beenisolated from an organism which naturally produces the enzyme as anative enzyme. The term “obtained” also means herein that the enzyme mayhave been produced recombinantly in a host organism, wherein therecombinantly produced enzyme is either native or foreign to the hostorganism or has a modified amino acid sequence, e.g., having one or moreamino acids which are deleted, inserted and/or substituted, i.e., arecombinantly produced enzyme which is a mutant and/or a fragment of anative amino acid sequence or an enzyme produced by nucleic acidshuffling processes known in the art. Encompassed within the meaning ofa native enzyme are natural variants and within the meaning of a foreignenzyme are variants obtained recombinantly, such as by site-directedmutagenesis or shuffling.

The enzymes may also be purified. The term “purified” as used hereincovers enzymes free from other components from the organism from whichit is derived. The term “purified” also covers enzymes free fromcomponents from the native organism from which it is obtained. Theenzymes may be purified, with only minor amounts of other proteins beingpresent. The expression “other proteins” relate in particular to otherenzymes. The term “purified” as used herein also refers to removal ofother components, particularly other proteins and most particularlyother enzymes present in the cell of origin of the enzyme of theinvention. The enzyme may be “substantially pure polypeptide,” that is,free from other components from the organism in which it is produced,that is, for example, a host organism for recombinantly producedenzymes.

The enzymes used in the present invention may be in any form suitablefor use in the processes described herein, such as, for example, a crudefermentation broth with or without cells, a dry powder or granulate, anon-dusting granulate, a liquid, a stabilized liquid, or a protectedenzyme. Granulates may be produced, e.g., as disclosed in U.S. Pat. Nos.4,106,991 and 4,661,452, and may optionally be coated by process knownin the art. Liquid enzyme preparations may, for instance, be stabilizedby adding stabilizers such as a sugar, a sugar alcohol or anotherpolyol, and/or lactic acid or another organic acid according toestablished process. Protected enzymes may be prepared according to theprocess disclosed in EP 238,216.

Hemicellulases

Enzymatic hydrolysis of hemicelluloses can be performed by a widevariety of fungi and bacteria. Similar to cellulose degradation,hemicellulose hydrolysis requires coordinated action of many enzymes.Hemicellulases can be placed into three general categories: theendo-acting enzymes that attack internal bonds within the polysaccharidechain, the exo-acting enzymes that act processively from either thereducing or nonreducing end of polysaccharide chain, and the accessoryenzymes, acetylesterases and esterases that hydrolyze lignin glycosidebonds, such as coumaric acid esterase and ferulic acid esterase (Wong,K. K. Y., Tan, L. U. L., and Saddler, J. N., 1988, Multiplicity ofβ-1,4-xylanase in microorganisms: Functions and applications, Microbiol.Rev. 52: 305-317; Tenkanen, M., and Poutanen, K., 1992, Significance ofesterases in the degradation of xylans, in Xylans and Xylanases, Visser,J., Beldman, G., Kuster-van Someren, M. A., and Voragen, A. G. J., eds.,Elsevier, New York, N.Y., 203-212; Coughlan, M. P., and Hazlewood, G.P., 1993, Hemicellulose and hemicellulases, Portland, London, UK;Brigham, J. S., Adney, W. S., and Himmel, M. E., 1996, Hemicellulases:Diversity and applications, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC,119-141).

Hemicellulases include xylanases, arabinofuranosidases, acetyl xylanesterase, glucuronidases, endo-galactanase, mannanases, endo or exoarabinases, exo-galactanses, and mixtures thereof. Examples ofendo-acting hemicellulases and ancillary enzymes includeendoarabinanase, endoarabinogalactanase, endoglucanase, endomannanase,endoxylanase, and feraxan endoxylanase. Examples of exo-actinghemicellulases and ancillary enzymes include α-L-arabinosidase,β-L-arabinosidase, α-1,2-L-fucosidase, α-D-galactosidase,β-D-galactosidase, β-D-glucosidase, β-D-glucuronidase, β-D-mannosidase,β-D-xylosidase, exoglucosidase, exocellobiohydrolase,exomannobiohydrolase, exomannanase, exoxylanase, xylan α-glucuronidase,and coniferin β-glucosidase. Examples of esterases include acetylesterases (acetylgalactan esterase, acetylmannan esterase, andacetylxylan esterase) and aryl esterases (coumaric acid esterase andferulic acid esterase).

Preferably, the hemicellulase is an exo-acting hemicellulase, and morepreferably, an exo-acting hemicellulase which has the ability tohydrolyze hemicellulose under acidic conditions of below pH 7. Anexample of a hemicellulase suitable for use in the present inventionincludes VISCOZYME™ (available from Novozymes A/S, Denmark). Thehemicellulase is added in an effective amount from about 0.001% to about5.0% wt. of solids, more preferably from about 0.025% to about 4.0% wt.of solids, and most preferably from about 0.005% to about 2.0% wt. ofsolids.

A xylanase (E.C. 3.2.1.8) may be obtained from any suitable source,including fungal and bacterial organisms, such as Aspergillus,Disporotrichum, Penicillium, Neurospora, Fusarium, Trichoderma,Humicola, Thermomyces, and Bacillus. Preferred commercially availablepreparations comprising xylanase include SHEARZYME®, BIOFEED WHEAT®,BIO-FEED Plus® L, CELLUCLAST®, ULTRAFLO®, VISCOZYME®, PENTOPAN MONO® BG,and PULPZYME® HC (Novozymes A/S); and LAMINEX® and SPEZYME® CP (GenencorInt.).

Esterases

Esterases that can be used for bioconversion of cellulose include acetylesterases such as acetylgalactan esterase, acetylmannan esterase, andacetylxylan esterase, and esterases that hydrolyze lignin glycosidebonds, such as coumaric acid esterase and ferulic acid esterase.

As used herein, an “esterase” also known as a carboxylic esterhydrolyase, refers to enzymes acting on ester bonds, and includesenzymes classified in EC 3.1.1 Carboxylic Ester Hydrolases according toEnzyme Nomenclature (Enzyme Nomenclature, 1992, Academic Press, SanDiego, Calif., with Supplement 1 (1993), Supplement 2 (1994), Supplement3 (1995), Supplement 4 (1997) and Supplement 5, in Eur. J. Biochem. 223:1-5, 1994; Eur. J. Biochem. 232: 1-6, 1995; Eur. J. Biochem. 237: 1-5,1996; Eur. J. Biochem. 250: 1-6, 1997, and Eur. J. Biochem. 264:610-650, 1999; respectively). Non-limiting examples of esterases includearylesterase, triacylglycerol lipase, acetylesterase,acetylcholinesterase, cholinesterase, tropinesterase, pectinesterase,sterol esterase, chlorophyllase, L-arabinonolactonase, gluconolactonase,uronolactonase, tannase, retinyl-palmitate esterase,hydroxybutyrate-dimer hydrolase, acylglycerol lipase, 3-oxoadipateenol-lactonase, 1,4-lactonase, galactolipase, 4-pyridoxolactonase,acylcarnitine hydrolase, aminoacyl-tRNA hydrolase, D-arabinonolactonase,6-phosphogluconolactonase, phospholipase A1,6-acetylglucose deacetylase,lipoprotein lipase, dihydrocoumarin lipase, limonin-D-ring-lactonase,steroid-lactonase, triacetate-lactonase, actinomycin lactonase,orsellinate-depside hydrolase, cephalosporin-C deacetylase, chlorogenatehydrolase, alpha-amino-acid esterase, 4-methyloxaloacetate esterase,carboxymethylenebutenolidase, deoxylimonate A-ring-lactonase,2-acetyl-1-alkylglycerophosphocholine esterase, fusarinine-Cornithinesterase, sinapine esterase, wax-ester hydrolase,phorbol-diester hydrolase, phosphatidylinositol deacylase, sialateO-acetylesterase, acetoxybutynylbithiophene deacetylase,acetylsalicylate deacetylase, methylumbelliferyl-acetate deacetylase,2-pyrone-4,6-dicarboxylate lactonase, N-acetylgalactosaminoglycandeacetylase, juvenile-hormone esterase, bis(2-ethylhexyl)phthalateesterase, protein-glutamate methylesterase, 11-cis-retinyl-palmitatehydrolase, all-trans-retinyl-palmitate hydrolase,L-rhamnono-1,4-lactonase, 5-(3,4-diacetoxybut-1-ynyl)-2,2′-bithiophenedeacetylase, fatty-acyl-ethyl-ester synthase, xylono-1,4-lactonase,N-acetylglucosaminylphosphatidylinositol deacetylase, cetraxatebenzylesterase, acetylalkylglycerol acetylhydrolase, and acetylxylanesterase.

Preferred esterases for use in the present invention are lipolyticenzymes, such as, lipases (classified as EC 3.1.1.3, EC 3.1.1.23, and/orEC 3.1.1.26) and phospholipases (classified as EC 3.1.1.4 and/or EC3.1.1.32, including lysophospholipases classified as EC 3.1.1.5). Otherpreferred esterases are cutinases (classified as EC 3.1.1.74).

The esterase may be added in an amount effective to obtain the desiredbenefit to improve the performance of the fermenting microorganism, forexample, to change the lipid composition/concentration inside and/oroutside of the fermenting microorganism or in the cell membrane of thefermenting microorganism, to result in an improvement in the movement ofsolutes into and/or out of the fermenting microorganisms duringfermentation and/or to provide more metabolizable energy sources (suchas, for example, by converting components, such as, oil from the cornsubstrate, to components useful the fermenting microorganism, e.g.,unsaturated fatty acids and glycerol), to increase ethanol yield.Examples of effective amounts of esterase are from about 0.01 to about400 LU/g DS (Dry Solids). Preferably, the esterase is used in an amountof about 0.1 to about 100 LU/g DS, more preferably about 0.5 to about 50LU/g DS, and even more preferably about 1 to about 20 LU/g DS. Furtheroptimization of the amount of esterase can hereafter be obtained usingstandard procedures known in the art.

One Lipase Unit (LU) is the amount of enzyme which liberates 1.0 μmol oftitratable fatty acid per minute with tributyrin as substrate and gumarabic as an emulsifier at 30(C, pH 7.0 (phosphate buffer).

In a preferred aspect, the esterase is a lipolytic enzyme, morepreferably, a lipase. As used herein, a “lipolytic enzyme” refers tolipases and phospholipases (including lyso-phospholipases). Thelipolytic enzyme is preferably of microbial origin, in particular ofbacterial, fungal or yeast origin. The lipolytic enzyme used may bederived from any source, including, for example, a strain of Absidia, inparticular Absidia blakesleena and Absidia corymbifera, a strain ofAchromobacter, in particular Achromobacter iophagus, a strain ofAeromonas, a strain of Alternaria, in particular Alternaria brassiciola,a strain of Aspergillus, in particular Aspergillus niger, Aspergillusoryzae, Aspergillus fumigatus, and Aspergillus flavus, a strain ofAchromobacter, in particular Achromobacter iophagus, a strain ofAureobasidium, in particular Aureobasidium pullulans, a strain ofBacillus, in particular Bacillus pumilus, Bacillus stearothermophilus,and Bacillus subtilis, a strain of Beauveria, a strain of Brochothrix,in particular Brochothrix thermosohata, a strain of Candida, inparticular Candida cylindracea (Candida rugosa), Candida paralipolytica,and Candida antarctica, a strain of Chromobacter, in particularChromobacter viscosum, a strain of Coprinus, in particular Coprinuscinereus, a strain of Fusarium, in particular Fusarium graminearum,Fusarium oxysporum, Fusarium solani, Fusarium solani pisi, Fusariumroseum culmorum, and Fusarium venenatum, a strain of Geotricum, inparticular Geotricum penicillatum, a strain of Hansenula, in particularHansenula anomala, a strain of Humicola, in particular Humicolabrevispora, Humicola brevis var. thermoidea, and Humicola insolens, astrain of Hyphozyma, a strain of Lactobacillus, in particularLactobacillus curvatus, a strain of Metarhizium, a strain of Mucor, astrain of Paecilomyces, a strain of Penicillium, in particularPenicillium cyclopium, Penicillium crustosum and Penicillium expansum, astrain of Pseudomonas in particular Pseudomonas aeruginosa, Pseudomonasalcaligenes, Pseudomonas cepacia (syn. Burkholderia cepacia),Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas maltophilia,Pseudomonas mendocina, Pseudomonas mephitica lipolytica, Pseudomonasalcaligenes, Pseudomonas plantari, Pseudomonas pseudoalcaligenes,Pseudomonas putida, Pseudomonas stutzeri, and Pseudomonaswisconsinensis, a strain of Rhizooctonia, in particular Rhizooctoniasolani, a strain of Rhizomucor, in particular Rhizomucor miehei, astrain of Rhizopus, in particular Rhizopus japonicus, Rhizopusmicrosporus, and Rhizopus nodosus, a strain of Rhodosporidium, inparticular Rhodosporidium toruloides, a strain of Rhodotorula, inparticular Rhodotorula glutinis, a strain of Sporobolomyces, inparticular Sporobolomyces shibatanus, a strain of Thermomyces, inparticular Thermomyces lanuginosus (formerly Humicola lanuginosa), astrain of Thiarosporella, in particular Thiarosporella phaseolina, astrain of Trichoderma, in particular, Trichoderma harzianum andTrichoderma reesei, and/or a strain of Verticillium.

In a preferred aspect, the lipolytic enzyme is derived from a strain ofAspergillus, Achromobacter, Bacillus, Candida, Chromobacter, Fusarium,Humicola, Hyphozyma, Pseudomonas, Rhizomucor, Rhizopus, or Thermomyces.

In more preferred aspects, the lipolytic enzyme is a lipase. Lipases maybe applied herein for their ability to modify the structure andcomposition of triglyceride oils and fats in the fermentation media(including fermentation yeast), for example, resulting from a cornsubstrate. Lipases catalyze different types of triglyceride conversions,such as hydrolysis, esterification, and transesterification. Suitablelipases include acidic, neutral, and basic lipases, as are well-known inthe art, although acidic lipases (such as, e.g., the lipase G AMANO 50,available from Amano) appear to be more effective at lowerconcentrations of lipase as compared to either neutral or basic lipases.Preferred lipases for use in the present invention include Candidaantarcitca lipase and Candida cylindracea lipase. More preferred lipasesare purified lipases such as Candida antarcitca lipase (lipase A),Candida antarcitca lipase (lipase B), Candida cylindracea lipase, andPenicillium camembertii lipase.

The lipase may be the one disclosed in EP 258,068-A or may be a lipasevariant such as a variant disclosed in WO 00/60063 or WO 00/32758,hereby incorporated by reference.

Lipases are preferably added in amounts from about 1 to about 400 LU/gDS, preferably about 1 to about 10 LU/g DS, and more preferably about 1to about 5 LU/g DS.

In another preferred aspect, the esterase is a cutinase. Cutinases areenzymes which are able to degrade cutin. The cutinase may be derivedfrom any source. In a preferred aspect, the cutinase is derived from astrain of Aspergillus, in particular Aspergillus oryzae, a strain ofAlternaria, in particular Alternaria brassiciola, a strain of Fusarium,in particular Fusarium solani, Fusarium solani pisi, Fusarium roseumculmorum, or Fusarium roseum sambucium, a strain of Helminthosporum, inparticular Helminthosporum sativum, a strain of Humicola, in particularHumicola insolens, a strain of Pseudomonas, in particular Pseudomonasmendocina or Pseudomonas putida, a strain of Rhizooctonia, in particularRhizooctonia solani, a strain of Streptomyces, in particularStreptomyces scabies, or a strain of Ulocladium, in particularUlocladium consortiale. In a most preferred aspect the cutinase isderived from a strain of Humicola insolens, in particular the strainHumicola insolens DSM 1800. Humicola insolens cutinase is described inWO 96/13580, which is hereby incorporated by reference. The cutinase maybe a variant such as one of the variants disclosed in WO 00/34450 and WO01/92502, which are hereby incorporated by reference. Preferred cutinasevariants include variants listed in Example 2 of WO 01/92502 which arehereby specifically incorporated by reference. An effective amount ofcutinase is from about 0.01 to about 400 LU/g DS, preferably from about0.1 to about 100 LU/g DS, and more preferably from about 1 to about 50LU/g DS. Further optimization of the amount of cutinase can hereafter beobtained using standard procedures known in the art.

In another preferred aspect, the esterase is a phospholipase. As usedherein, the term “phospholipase” is an enzyme which has activity towardsphospholipids, e.g., hydrolytic activity. Phospholipids, such aslecithin or phosphatidylcholine, consist of glycerol esterified with twofatty acids in an outer (sn-1) and the middle (sn-2) positions andesterified with phosphoric acid in the third position. The phosphoricacid may be esterified to an amino-alcohol. Several types ofphospholipase activity can be distinguished, including phospholipases A1and A2 which hydrolyze one fatty acyl group (in the sn-1 and sn-2position, respectively) to form lysophospholipid; and lysophospholipase(or phospholipase B) which hydrolyzes the remaining fatty acyl group inlysophospholipid. Phospholipase C and phospholipase D(phosphodiesterases) release diacyl glycerol or phosphatidic acidrespectively.

The term “phospholipase” includes enzymes with phospholipase activity,e.g., phospholipase A (A1 or A2), phospholipase B activity,phospholipase C activity, or phospholipase D activity. The term“phospholipase A” as used herein is intended to cover an enzyme withphospholipase A1 and/or phospholipase A2 activity. The phospholipaseactivity may be provided by enzymes having other activities as well,such as, e.g., a lipase with phospholipase activity. The phospholipaseactivity may, for example, be from a lipase with phospholipase sideactivity. In other aspects, the phospholipase enzyme activity isprovided by an enzyme having essentially only phospholipase activity andwherein the phospholipase enzyme activity is not a side activity.

The phospholipase may be of any origin, for example, of animal origin(e.g., mammalian, for example, bovine or porcine pancreas), or snakevenom or bee venom. Alternatively, the phospholipase may be of microbialorigin, for example, from filamentous fungi, yeast or bacteria, such asAspergillus, e.g., A. awamori, A. foetidus, A. japonicus, A. niger, orA. oryzae, Dictyostelium, e.g., D. discoideum; Fusarium, e.g., F.culmorum, F. graminearum, F. heterosporum, F. solani, F. oxysporum, orF. venenatum; Mucor, e.g., M. javanicus, M. mucedo, or M. subtilissimus;Neurospora, e.g., N. crassa; Rhizomucor, e.g., R. pusillus; Rhizopus,e.g., R. arrhizus, R. japonicus, or R. stolonifer; Sclerotinia, e.g., S.libertiana; Trichophyton, e.g., T. rubrum; Whetzelinia, e.g., W.sclerotiorum; Bacillus, e.g., B. megaterium or B. subtilis; Citrobacter,e.g., C. freundii; Enterobacter, e.g., E. aerogenes or E. cloacae;Edwardsiella, E. tarda; Erwinia, e.g., E. herbicola; Escherichia, e.g.,E. coli; Klebsiella, e.g., K. pneumoniae; Proteus, e.g., P. vulgaris;Providencia, e.g., P. stuartii, Salmonella, e.g., S. typhimurium;Serratia, e.g., S. liquefasciens, S. marcescens; Shigella, e.g., S.flexneri; Streptomyces, e.g., S. violeceoruber; or Yersinia, e.g., Y.enterocolitica.

Preferred commercial phospholipases include LECITASE™ and LECITASE™ULTRA (available from Novozymes A/S, Denmark).

An effective amount of phospholipase is from about 0.01 to about 400LU/g DS, preferably from about 0.1 to about 100 LU/g DS, and morepreferably from about 1 to about 50 LU/g DS. Further optimization of theamount of phospholipase can hereafter be obtained using standardprocedures known in the art.

Proteases

In another preferred aspect of the invention, at least one surfactantand at least one carbohydrate generating enzyme is used in combinationwith at least one protease. The protease may be used, e.g., to digestprotein to produce free amino nitrogen (FAN). Such free amino acidsfunction as nutrients for the yeast, thereby enhancing the growth of theyeast and, consequently, the production of ethanol.

The fermenting microorganism for use in a fermentation process may beproduced by propagating the fermenting microorganism in the presence ofat least one protease. Although not limited to any one theory ofoperation, it is believed that the propagation of the fermentingmicroorganism with an effective amount of at least one protease reducesthe lag time of the fermenting microorganism when the fermentingmicroorganism is subsequently used in a fermentation process as comparedto a fermenting microorganism that was propogated under the sameconditions without the addition of the protease. The action of theprotease in the propagation process is believed to directly orindirectly result in the suppression or expression of genes which aredetrimental or beneficial, respectively, to the fermenting microorganismduring fermentation, thereby decreasing lag time and resulting in afaster fermentation cycle.

Proteases are well known in the art and refer to enzymes that catalyzethe cleavage of peptide bonds. Suitable proteases include fungal andbacterial proteases. Preferred proteases are acidic proteases, i.e.,proteases characterized by the ability to hydrolyze proteins underacidic conditions below pH 7. Suitable acid fungal proteases includefungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida,Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotium, andTorulopsis. Especially contemplated are proteases derived fromAspergillus niger (see, e.g., Koaze et. al., 1964, Agr. Biol. Chem.Japan 28: 216), Aspergillus saitoi (see, e.g., Yoshida, 1954, J. Agr.Chem. Soc. Japan 28: 66), Aspergillus awamori (Hayashida et al., 1977,Agric. Biol. Chem. 42: 927-933, Aspergillus aculeatus (WO 95/02044), orAspergillus oryzae; and acidic proteases from Mucor pusillus or Mucormiehei.

Bacterial proteases, which are not acidic proteases, include thecommercially available products ALCALASE™ and NEUTRASE™ (available fromNovozymes A/S). Other proteases include GC106 from GenencorInternational, Inc., USA and NOVOZYM™ 50006 from Novozymes A/S.

Preferably, the protease is an aspartic acid protease, as described, forexample, in Handbook of Proteolytic Enzymes, Edited by A. J. Barrett, N.D. Rawlings and J. F. Woessner, Academic Press, San Diego, 1998, Chapter270). Suitable examples of aspartic acid protease include, e.g., thosedisclosed by Berka et al., 1990, Gene 96: 313; Berka et al., 1993, Gene125: 195-198; and Gomi et al., 1993, Biosci. Biotech. Biochem. 57:1095-1100.

Peroxidases

Other compounds possessing peroxidase activity may be any peroxidase (EC1.11.1.7), or any fragment having peroxidase activity derived therefrom,exhibiting peroxidase activity.

Preferably, the peroxidase is produced by plants (e.g., horseradish orsoybean peroxidase) or microorganisms such as fungi or bacteria.

Some preferred fungi include strains belonging to the subdivisionDeuteromycotina, class Hyphomycetes, e.g., Fusarium, Humicola,Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces,Ulocladium, Embellisia, Cladosporium, or Dreschlera, in particular,Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma reesei,Myrothecium verrucaria (IFO 6113), Verticillum alboatrum, Verticillumdahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago,Ulocladium chartarum, Embellisia alli, or Dreschlera halodes.

Other preferred fungi include strains belonging to the subdivisionBasidiomycotina, class Basidiomycetes, e.g., Coprinus, Phanerochaete,Coriolus, or Trametes, in particular Coprinus cinereus f. microsporus(IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g.NA-12), or Trametes (previously called Polyporus), e.g., T. versicolor(e.g., PR4 28-A).

Further preferred fungi include strains belonging to the subdivisionZygomycotina, class Mycoraceae, e.g., Rhizopus or Mucor, in particularMucor hiemalis.

Some preferred bacteria include strains of the order Actinomycetales,e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus(IFO 12382), or Streptoverticillum verticillium ssp. verticillium.

Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonaspalustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958),Pseudomonas fluorescens (NRRL B-11), and Bacillus strains, e.g.,Bacillus pumilus (ATCC 12905) and Bacillus stearothermophilus.

Further preferred bacteria include strains belonging to Myxococcus,e.g., M. virescens.

The peroxidase may also be one which is produced by a method comprisingcultivating a host cell transformed with a recombinant DNA vector whichcarries a DNA sequence encoding the peroxidase as well as DNA sequencesfor expression of the DNA sequence encoding the peroxidase, in a culturemedium under conditions permitting the expression of the peroxidase andrecovering the peroxidase from the culture.

In a preferred aspect, a recombinantly produced peroxidase is aperoxidase derived from a Coprinus sp., in particular C. macrorhizus orC. cinereus according to WO 92/16634.

In the present invention, compounds possessing peroxidase activitycomprise peroxidase enzymes and peroxidase active fragments derived fromcytochromes, hemoglobin, or peroxidase enzymes.

One peroxidase unit (POXU) is the amount of enzyme which under thefollowing conditions catalyzes the conversion of 1 μmole hydrogenperoxide per minute at 30° C. in 0.1 M phosphate buffer pH 7.0, 0.88 mMhydrogen peroxide, and 1.67 mM2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). The reactionis followed for 60 seconds (15 seconds after mixing) by the change inabsorbance at 418 nm, which should be in the range of 0.15 to 0.30. Forcalculation of activity, an absorption coefficient of oxidized ABTS of36 mM-1 cm-1 and a stoichiometry of one μmole H₂O₂ converted per twoμmole ABTS oxidized are used.

Laccases

In the present invention, laccases and laccase related enzymes compriseany laccase enzyme classified as EC 1.10.3.2, any catechol oxidaseenzyme classified as EC 1.10.3.1, any bilirubin oxidase enzymeclassified as EC 1.3.3.5, or any monophenol monooxygenase enzymeclassified as EC 1.14.18.1.

The above-mentioned enzymes may be microbial, i.e., obtained frombacteria or fungi (including filamentous fungi and yeasts), or they maybe derived from plants.

Suitable examples from fungi include a laccase obtained from a strain ofAspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia,Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T.versicolor, Rhizooctonia, e.g., R. solani, Coprinus, e.g., C. cinereus,C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P.condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M.thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P.pinsitus, Pycnoporus, e.g., P. cinnabarinus, Phlebia, e.g., P. radita(WO 92/01046), or Coriolus, e.g., C. hirsutus (JP 2-238885).

Suitable examples from bacteria include a laccase obtained from a strainof Bacillus.

A laccase obtained from Coprinus, Myceliophthora, Polyporus, Pycnoporus,Scytalidium or Rhizoctonia is preferred; in particular a laccaseobtained from Coprinus cinereus, Myceliophthora thermophila, Polyporuspinsitus, Pycnoporus cinnabarinus, Scytalidium thermophilum, orRhizoctonia solani.

Commercially available laccases are NS51001 (a Polyporus pinsitiuslaccase, available from Novozymes A/S, Denmark) and NS51002 (aMyceliopthora thermophila laccase, available from Novozymes A/S,Denmark).

The laccase or the laccase related enzyme may also be one which isproduced by a method comprising cultivating a host cell transformed witha recombinant DNA vector which carries a DNA sequence encoding thelaccase as well as DNA sequences for expression of the DNA sequenceencoding the laccase, in a culture medium under conditions permittingthe expression of the laccase enzyme, and recovering the laccase fromthe culture.

Laccase activity (LACU) is determined from the oxidation ofsyringaldazine under aerobic conditions at pH 5.5. The violet colorproduced is measured at 530 nm. The analytical conditions are 19 mMsyringaldazine, 23 mM acetate buffer, pH 5.5, 30° C., 1 minute reactiontime. One laccase unit (LACU) is the amount of enzyme that catalyses theconversion of 1.0 μmole syringaldazine per minute under the aboveconditions.

Laccase activity (LAMU) is determined from the oxidation ofsyringaldazine under aerobic conditions at pH 7.5. The violet colorproduced is photometered at 530 nm. The analytical conditions are 19 mMsyringaldazine, 23 mM Tris/maleate pH 7.5, 30° C., 1 minute reactiontime. One laccase unit (LAMU) is the amount of enzyme that catalyses theconversion of 1.0 μmole syringaldazine per minute under the aboveconditions.

The polypeptides having cellulolytic enhancing activity may be used inconjunction with the above-noted enzymes and/or cellulolytic proteins tofurther degrade the cellulose component of the biomass substrate, (see,for example, Brigham et al., 1995, in Handbook on Bioethanol (Charles E.Wyman, editor), pp. 119-141, Taylor & Francis, Washington D.C.; Lee,1997, Journal of Biotechnology 56: 1-24).

The optimum amounts of a polypeptide having cellulolytic enhancingactivity and of cellulolytic proteins depends on several factorsincluding, but not limited to, the mixture of component cellulolyticproteins, the cellulosic substrate, the concentration of cellulosicsubstrate, the pretreatment(s) of the cellulosic substrate, temperature,time, pH, and inclusion of fermenting organism (e.g., yeast forSimultaneous Saccharification and Fermentation).

In a preferred aspect, an effective amount of polypeptide(s) havingcellulolytic enhancing activity to cellulosic material is about 0.01 toabout 2.0 mg, preferably at about 0.025 to about 1.5 mg, more preferablyat about 0.05 to about 1.25 mg, more preferably at about 0.075 to about1.25 mg, more preferably at about 0.1 to about 1.25 mg, even morepreferably at about 0.15 to about 1.25 mg, and most preferably at about0.25 to about 1.0 mg per g of cellulosic material.

In another preferred aspect, an effective amount of cellulolyticprotein(s) to cellulosic material is about 0.5 to about 50 mg,preferably at about 0.5 to about 40 mg, more preferably at about 0.5 toabout 25 mg, more preferably at about 0.75 to about 20 mg, morepreferably at about 0.75 to about 15 mg, even more preferably at about0.5 to about 10 mg, and most preferably at about 2.5 to about 10 mg perg of cellulosic material.

In a preferred aspect, an effective amount of polypeptide(s) havingcellulolytic enhancing activity to cellulolytic protein(s) is about0.005 to about 1.0 g, preferably at about 0.01 to about 1.0 g, morepreferably at about 0.15 to about 0.75 g, more preferably at about 0.15to about 0.5 g, more preferably at about 0.1 to about 0.5 g, even morepreferably at about 0.1 to about 0.5 g, and most preferably at about0.05 to about 0.2 g per g of cellulolytic protein(s).

Detergent Compositions

The polypeptides having cellulolytic enhancing activity may be added toand thus become a component of a detergent composition.

The detergent composition of the present invention may be formulated,for example, as a hand or machine laundry detergent compositionincluding a laundry additive composition suitable for pre-treatment ofstained fabrics and a rinse added fabric softener composition, or beformulated as a detergent composition for use in general household hardsurface cleaning operations, or be formulated for hand or machinedishwashing operations.

In a specific aspect, the present invention provides a detergentadditive comprising a polypeptide having cellulolytic enhancing activityas described herein. The detergent additive as well as the detergentcomposition may comprise one or more enzymes such as a protease, lipase,cutinase, an amylase, carbohydrase, cellulase, pectinase, mannanase,arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/orperoxidase.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metalloprotease, preferably an alkaline microbial proteaseor a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and274.

Preferred commercially available protease enzymes include Alcalase™,Savinase™, Primase™, Duralase™, Esperase™, and Kannase™ (Novozymes A/S),Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OXP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta,1131: 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include Lipolase™,Lipex™, and Lipolase Ultra™ (Novozymes A/S).

Amylases: Suitable amylases (alpha and/or beta) include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, α-amylases obtainedfrom Bacillus, e.g., a special strain of Bacillus licheniformis,described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (Novozymes A/S), and Rapidase™ and Purastar™ (from GenencorInternational Inc.).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

A detergent composition of the present invention may be in anyconvenient form, e.g., a bar, a tablet, a powder, a granule, a paste ora liquid. A liquid detergent may be aqueous, typically containing up to70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates, or layered silicates (e.g., SKS-6 fromHoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers,and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example, WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions, any enzyme may be added in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.05-5 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor.

In the detergent compositions, a polypeptide having cellulolyticenhancing activity may be added in an amount corresponding to 0.001-100mg of protein, preferably 0.005-50 mg of protein, more preferably0.01-25 mg of protein, even more preferably 0.05-10 mg of protein, mostpreferably 0.05-5 mg of protein, and even most preferably 0.01-1 mg ofprotein per liter of wash liquor.

A polypeptide of the invention having cellulolytic enhancing activitymay also be incorporated in the detergent formulations disclosed in WO97/07202, which is hereby incorporated by reference.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Trichoderma reesei RutC30 (ATCC 56765; Montenecourt and Eveleigh, 1979,Adv. Chem. Ser. 181: 289-301) was used as the source of a Trichodermareesei gene encoding a Family 61 polypeptide having cellulolyticenhancing activity. Aspergillus oryzae JaL250 strain (WO 99/61651) wasused for expression of the Trichoderm reesei Family 61 polypeptidehaving cellulolytic enhancing activity.

Media

PDA plates were composed per liter of 39 grams of potato dextrose agar.

MDU2BP medium was composed per liter of 45 g of maltose, 1 g ofMgSO₄.7H₂O, 1 g of NaCl, 2 g of K₂SO₄, 12 g of KH₂PO₄, 7 g of yeastextract, 2 g of urea, and 0.5 ml of AMG trace metals solution, pH to5.0.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g ofFeSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, and 3 g of citric acid.

LB plates were composed per liter of 10 g of tryptone, 5 g of yeastextract, 5 g of sodium chloride, and 15 g of Bacto Agar.

SOC medium was composed of 2% tryptone, 0.5% yeast extract, 10 mM NaCl,2.5 mM KCl, 10 mM MgCl₂, and 10 mM MgSO₄, and filter-sterilized glucoseto 20 mM added after autoclaving.

Example 1 Fermentation and Mycelial Tissue

Trichoderma reesei strain RutC30 was cultivated in a pilot scalefermentation tank in growth medium containing a complex carbon source.The carbon sources included glucose, cellulose, or pre-treated andwashed corn stover. Fungal mycelium was collected from a one-litersample, and immediately frozen in liquid N₂ and stored at −80° C.

Pretreated corn stover (PCS) was obtained from the U.S. Department ofEnergy National Renewable Energy Laboratory (NREL). The water-insolublesolids in PCS are 56.5% cellulose, 4.6% hemicellulose, and 28.4% lignin.Pretreatment conditions were corn stover, 1.4% (wt/vol) sulfuric acid,165° C., 107 psi, for 8 minutes. Prior to assay, PCS was washed with alarge volume of distilled deionized water on a glass filter. PCS wasthen milled using a coffee grinder to reduce particle size, and washedfurther with water on a 22 μm Millipore filter (6P Express Membrane,Stericup, Millipore, Billerica, Mass.). The washed PCS was resuspendedin deionized water to make a 20 mg/ml suspension, and stored at 4° C.

Example 2 Trichoderma reesei Directional cDNA Library Construction

Total RNA was prepared from the Trichoderma reesei mycelial samplesdescribed in Example 1 by extraction with guanidinium thiocyanatefollowed by ultracentrifugation through a 5.7 M CsCl cushion (Chirgwinet al., 1979, Biochemistry 18: 5294-5299) using the followingmodifications. The frozen mycelia were ground in liquid N₂ to a finepowder with a mortar and a pestle, followed by grinding in a precooledcoffee mill, and immediately suspended in 5 volumes of RNA extractionbuffer (4 M guanidinium thiocyanate, 0.5% sodium laurylsarcosine, 25 mMsodium citrate pH 7.0, 0.1 M β-mercaptoethanol). The mixture was stirredfor 30 minutes at room temperature and centrifuged (20 minutes at12,000×g) to pellet the cell debris. The supernatant was collected,carefully layered onto a 5.7 M CsCl cushion (5.7 M CsCl, 10 mM EDTA, pH7.5, 0.1% diethylpyrocarbonate (DEPC); autoclaved prior to use) using26.5 ml of supernatant per 12.0 ml of CsCl cushion, and centrifuged toobtain the total RNA (Beckman SW 28 rotor, 25,000 rpm, room temperature,24 hours). After centrifugation the supernatant was carefully removedand the bottom of the tube containing the RNA pellet was cut off andrinsed with 70% ethanol. The total RNA pellet was transferred to anEppendorf tube, suspended in 500 μl of TE (10 mM Tris-0.1 mM EDTA), pH7.6 (if difficult, heated occasionally for 5 minutes at 65° C.), phenolextracted, and precipitated with ethanol for 12 hours at −20° C. (2.5volumes of ethanol, 0.1 volume of 3M sodium acetate pH 5.2). The RNA wascollected by centrifugation (30 minutes at 12,000×g), washed in 70%ethanol, and resuspended in a minimum volume of DEPC-treated water. Thetotal RNA concentration was determined by measuring the absorbance at260 nm.

Poly(A)⁺ RNA was isolated by oligo(dT)-cellulose affinity chromatography(Aviv & Leder, 1972, Proceedings of the National Academy of Sciences USA69: 1408-1412). A total of 0.2 g of oligo(dT) cellulose (BoehringerMannheim, Indianapolis, Ind.) was pre-swollen in 10 ml of 1× of columnloading buffer (20 mM Tris-Cl, pH 7.6, 0.5 M NaCl, 1 mM EDTA, 0.1% SDS),loaded onto a DEPC-treated, plugged plastic column (Poly PrepChromatography Column, BioRad, Hercules, Calif.), and equilibrated with20 ml of 1× loading buffer. The total RNA (1-2 mg) was heated at 65° C.for 8 minutes, quenched on ice for 5 minutes, and after addition of 1volume of 2× column loading buffer loaded onto the column. The eluatewas collected and reloaded 2-3 times by heating the sample as above andquenching on ice prior to each loading. The oligo(dT) column was washedwith 10 volumes of 1×0 loading buffer, then with 3 volumes of mediumsalt buffer (20 mM Tris-Cl, pH 7.6, 0.1 M NaCl, 1 mM EDTA, 0.1% SDS),followed by elution of the poly(A)⁺ RNA with 3 volumes of elution buffer(10 mM Tris-Cl pH 7.6, 1 mM EDTA, 0.05% SDS) preheated to 65° C., bycollecting 500 μl fractions. The absorbance at 260 nm was read for eachcollected fraction, and the mRNA containing fractions were pooled andethanol precipitated at −20° C. for 12 hours. The poly(A)⁺ RNA wascollected by centrifugation, resuspended in DEPC-treated water, andstored in 5-10 μg aliquots at −80° C.

Double-stranded Eco RI-Not I-directional cDNA was synthesized from 5 μgof Trichoderma reesei RutC30 poly(A)⁺ RNA by the RNase H method (Gublerand Hoffman 1983, Gene 25: 263-270; Sambrook et al., 1989, MolecularCloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.) using a hair-pin modification. The poly(A)⁺ RNA (5μg in 5 μl of DEPC-treated water) was heated at 70° C. for 8 minutes ina pre-siliconized, RNase-free Eppendorf tube, quenched on ice, andcombined in a final volume of 50 μl with reverse transcriptase buffer(50 mM Tris-Cl pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT) containing 1 mMof dATP, dGTP and dTTP, 0.5 mM of 5-methyl-dCTP, 40 units of humanplacental ribonuclease inhibitor (Promega, Madison, Wis.), 4.81 μg ofoligo(dT)₁₈-Not I primer, and 1000 units of SuperScript II RNaseH—reverse transcriptase (Life Technologies, Inc., Rockville, Md.).First-strand cDNA was synthesized by incubating the reaction mixture at45° C. for 1 hour. After synthesis, the mRNA:cDNA hybrid mixture was gelfiltrated through a MicroSpin S-400 HR spin column (AmershamBiosciences, Piscataway, N.J.) according to the manufacturer'sinstructions.

After gel filtration, the hybrids were diluted in 250 μl of secondstrand buffer (20 mM Tris-Cl pH 7.4, 90 mM KCl, 4.6 mM MgCl₂, 10 mM(NH₄)₂SO₄, 0.16 mM NAD⁺) containing 200 μM of each dNTP, 60 units of E.coli DNA polymerase I (Amersham Biosciences, Piscataway, N.J.), 5.25units of RNase H, and 15 units of E. coli DNA ligase (New EnglandBiolabs, Inc., Beverly, Mass.). Second strand cDNA synthesis wasperformed by incubating the reaction tube at 16° C. for 2 hours, and anadditional 15 minutes at 25° C. The reaction was stopped by addition ofEDTA to 20 mM final concentration followed by phenol and chloroformextractions.

The double-stranded cDNA was ethanol precipitated at −20° C. for 12hours by addition of 2 volumes of 96% ethanol and 0.2 volume of 10 Mammonium acetate, recovered by centrifugation, washed in 70% ethanol,dried (SpeedVac), and resuspended in 30 μl of Mung bean nuclease buffer(30 mM sodium acetate pH 4.6, 300 mM NaCl, 1 mM ZnSO₄, 0.35 mMdithiothreitol, 2% glycerol) containing 25 units of Mung bean nuclease.The single-stranded hair-pin DNA was clipped by incubating the reactionat 30° C. for 30 minutes, followed by addition of 70 μl of 10 mMTris-Cl, pH 7.5, 1 mM EDTA, phenol extraction, and ethanol precipitationwith 2 volumes of 96% ethanol and 0.1 volume 3 M sodium acetate pH 5.2on ice for 30 minutes.

The double-stranded cDNAs were recovered by centrifugation (30,000×g for30 minutes), and blunt-ended with T4 DNA polymerase in 30 μl of T4 DNApolymerase buffer (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate,50 mM potassium acetate, 1 mM dithiothreitol) containing 0.5 mM of eachdNTP, and 5 units of T4 DNA polymerase by incubating the reactionmixture at 16° C. for 1 hour. The reaction was stopped by addition ofEDTA to 20 mM final concentration, followed by phenol and chloroformextractions and ethanol precipitation for 12 hours at −20° C. by adding2 volumes of 96% ethanol and 0.1 volume of 3 M sodium acetate pH 5.2.

After the fill-in reaction, cDNA was recovered by centrifugation asabove, washed in 70% ethanol, and the DNA pellet was dried in aSpeedVac. The cDNA pellet was resuspended in 25 μl of ligation buffer(30 mM Tris-Cl, pH 7.8, 10 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM ATP)containing 2 μg of Eco RI adaptors (0.2 μg/μl, Amersham Biosciences,Piscataway, N.J.) and 20 units of T4 ligase (Roche MolecularBiochemicals, Indianapolis, Ind.) by incubating the reaction mix at 16°C. for 12 hours. The reaction was stopped by heating at 65° C. for 20minutes, and then placed on ice for 5 minutes. The adapted cDNA wasdigested with Not I by addition of 20 μl of autoclaved water, 5 μl of10× Not I restriction enzyme buffer, and 50 units of Not I, followed byincubation for 3 hours at 37° C. The reaction was stopped by heating thesample at 65° C. for 15 minutes. The cDNAs were size-fractionated byagarose gel electrophoresis on a 0.8% SeaPlaque GTG low meltingtemperature agarose gel (FMC, Rockland, Me.) in 44 mM Tris Base, 44 mMboric acid, 0.5 mM EDTA (TBE) buffer (in autoclaved water) to separateunligated adaptors and small cDNAs. The gel was run for 12 hours at 15V, and the cDNA was size-selected with a cut-off at 0.7 kb by cuttingout the lower part of the agarose gel. Then a 1.5% agarose gel waspoured in front of the cDNA-containing gel, and the double-strandedcDNAs were concentrated by running the gel backwards until it appearedas a compressed band on the gel. The cDNA-containing gel piece was cutout from the gel and the cDNA was extracted from the gel using a GFX GelBand Purification Kit (Amersham, Arlington Heights, Ill.) as follows.The trimmed gel slice was weighed in a 2 ml nuclease-freemicrocentrifuge tube (ISC BioExpress, Kaysville, Utah). Then 10 ml ofCapture Buffer (Amersham, Arlington Heights, Ill.) was added for each 10mg of gel slice. The gel slice was dissolved by incubation at 60° C. for10 minutes, until the agarose was completely solubilized, and the samplewas then pelleted by a brief centrifugation (2 minutes at 8,000×g). Themelted sample was transferred to a GFX spin column placed in acollection tube, incubated at 25° C. for 1 minute, and then centrifugedat full speed (15,000×g) in a microcentrifuge for 30 seconds. Theflow-through was discarded, and the column was washed with 500 μl ofwash buffer (GFX Gel Band Purification Kit, Amersham, Arlington Heights,Ill.) followed by centrifugation at full speed for 30 seconds. Thecollection tube was discarded, and the column was placed in a 1.5 mlEppendorf tube, followed by elution of the cDNA by addition of 50 μl ofTE pH 7.5 to the center of the column, incubation at 25° C. for 1minute, and finally by centrifugation for 1 minute at maximum speed(15,000×g). The eluted cDNA was stored at −20° C. until libraryconstruction.

A plasmid DNA preparation for a Eco RI-Not I insert-containing pYES2.0cDNA clone was purified using a QIAGEN Tip-100 according to themanufacturer's instructions (QIAGEN, Valencia, Calif.). A total of 10 μgof purified plasmid DNA was digested to completion with Not I and Eco RIin a total volume of 60 μl by addition of 6 μl of 10× NEBuffer for EcoRI (New England Biolabs, Beverly, Mass.), 40 units of Not I, and 20units of Eco RI followed by incubation for 6 hours at 37° C. Thereaction was stopped by heating the sample at 65° C. for 20 minutes. Thedigested plasmid DNA was extracted once with phenol-chloroform, thenwith chloroform, followed by ethanol precipitation for 12 hours at −20°C. by adding 2 volumes of 96% ethanol and 0.1 volume of 3 M sodiumacetate pH 5.2. The precipitated DNA was resuspended in 25 μl of TE pH7.5, loaded onto a 0.8% SeaKem agarose gel in TBE buffer, and run for 3hours at 60 V. The digested vector was cut out from the gel, and the DNAwas extracted from the gel using a GFX Gel Band Purification Kitaccording to the manufacturer's instructions. After measuring the DNAconcentration by absorbance at 260 nm, the eluted vector was stored at−20° C. until library construction.

To establish the optimal ligation conditions for the cDNA library, fourtest ligations were performed in 10 μl of ligation buffer (30 mM Tris-ClpH 7.8, 10 mM MgCl₂, 10 mM DTT, 0.5 mM ATP) containing 7 μl ofdouble-stranded cDNA (corresponding to approximately 1/10 of the totalvolume in the cDNA sample), 2 units of T4 ligase, and 25 ng, 50 ng and75 ng of Eco RI-Not I cleaved pYES2.0 vector (Invitrogen, Carlsbad,Calif.), respectively. The vector background control ligation reactioncontained 75 ng of Eco RI-Not I cleaved pYES.0 vector without cDNA. Theligation reactions were performed by incubation at 16° C. for 12 hours,heated at 65° C. for 20 minutes, and then 10 μl of autoclaved water wasadded to each tube. One μl of the ligation mixtures was electroporated(200 W. 2.5 kV, 25 mF) to 40 μl of electrocompetent E. coli DH10B cells(Life Technologies, Gaithersburg, Md.). After addition of 1 ml of SOCmedium (Birren et al., 1998. Genome Analysis, Vol. 2. Detecting Genes.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) to eachtransformation mix, the cells were grown at 37° C. for 1 hour. Then 50μl and 5 μl from each electroporation were plated on LB platessupplemented with ampicillin at 100 μg per ml and grown at 37° C. for 12hours. Using the optimal conditions, a Trichoderma reesei RutC30 cDNAlibrary containing 1-2.5×10⁷ independent colony forming units wasestablished in E. coli, with a vector background of ca. 1%. The cDNAlibrary was stored as (1) individual pools (25,000 c.f.u./pool) in 20%glycerol at −80° C.; (2) cell pellets of the same pools at −20° C.; (3)QIAGEN Tip 100 purified plasmid DNA from individual pools at −20° C.;and (4) directional, double-stranded cDNA at −20° C.

Example 3 Trichoderma reesei EST Template Preparation

Plasmid DNAs from individual E. coli colonies from the cDNA librariesdescribed in Example 2 were purified using a 96-well manifold plasmidpreparation system (QIAGEN, Valencia, Calif.) according to instructionssupplied by the manufacturer.

Example 4 Analysis of DNA Sequence Data of cDNA Clones

Base calling, quality value assignment, and vector trimming wereperformed with the assistance of PHRED/PHRAP software (University ofWashington, Seattle, Wash.). Sequence homology analysis of the assembledEST sequences against the PIR database was performed with the Blastxprogram (Altschul et. al.,1990, J. Mol. Biol. 215:403-410) on a 32-nodeLinux cluster (Paracel, Inc., Pasadena, Calif.) using the BLOSUM 62matrix (Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919).

Example 5 Identification of a cDNA Clone Encoding a Family 61Polypeptide Having Cellulolytic Enhancing Activity (GH61B)

A cDNA clone encoding a Family 61 polypeptide having cellulolyticenhancing activity (GH61B) was initially identified by the similarity ofits translated product to other known Family 61 polypeptides present inpublic databases. Specifically, this analysis indicated that thepredicted polypeptide was 50% identical at the protein level to the coredomain of endoglucanase IV of Trichoderma reesei, a known member ofFamily 61 (Saloheimo et al., 1997, Eur. J. Biochem. 249: 584-591). Afterthis initial identification, the EST clone was retrieved from itoriginal frozen stock plate and streaked onto LB plates supplementedwith 100 μg of ampicillin per ml. The plates were incubated overnight at37° C. and the next day a single colony from each plate was used toinoculate 3 ml of LB supplemented with 100 μg of ampicillin per ml. Theliquid cultures were incubated overnight at 37° C. and plasmid DNA wasprepared with a BioRobot 9600 (QIAGEN Inc., Valencia, Calif.). PlasmidDNA from each EST clone was sequenced again with Big-Dye™ terminatorchemistry (Applied Biosystems, Inc., Foster City, Calif.), using the M13forward and a Poly-T primer shown below to sequence the 3′ end of theclone. (SEQ ID NO: 3) 5′-TTTTTTTTTTTTTTTTTTTTTTTVN-3′

where V=G, A, or C and N=G, A, C, or T

One clone, Trichoderma reesei EST Tr3337 designated pTr3337, wasselected for nucleotide sequence analysis.

Example 6 Characterization of a Trichoderma reesei cDNA Clone Encoding aPolypeptide Having Cellulolytic Enhancing Activity

Three new primers shown below were designed to extend the sequenceinformation from EST pTr3337 (FIG. 2). (SEQ ID NO: 4)5′-TGTCCATGGCCGAGA-3′ (SEQ ID NO: 5) 5′-ATACTGGTCACTTCCCCAA-3′ (SEQ IDNO: 6) 5′-GCGCTGGGTCAAGATT-3′

DNA sequencing was performed on a Perkin-Elmer Biosystems Model 377 XLAutomated DNA Sequencer using dye-terminator chemistry (Giesecke et al.,1992, Journal of Virology Methods 38: 47-60). The 1.17 kb cDNA fragmentwas sequenced to a Phred quality value of 36.

The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence(SEQ ID NO: 2) of the coding region of the Trichoderma reesei GH61B cDNAclone are shown in FIG. 1. The coding sequence is 750 bp including thestop codon. The encoded predicted protein is 249 amino acids. The codingregion is 56.8% G+C. Using the SignalP program (Nielsen et al., 1997,Protein Engineering 10: 1-6), a signal peptide of 19 residues waspredicted. The predicted mature protein contains 230 amino acids with amolecular mass of 25.1 kDa and isoelectric point (pl) of 7.36.

Analysis of the mature peptide with the Interproscan program (Zdobnovand Apweiler, 2001, Bioinformatics 17: 847-8) showed that the geneencoded by clone Tr3337 contained the sequence signature of the glycosylhydrolase Family 61 proteins. This sequence signature known as the Pfampattern PF03443 (Bateman et. al., 2004, Nucleic Acids Research 32:138-141) is located from residue 20 to 241 confirming that clone Tr3337encodes a Trichoderma reseei Family 61 gene.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theTrichoderma reesei gene encoding the GH61B mature polypeptide havingcellulolytic enhancing activity shared 100% and 62% identity (excludinggaps) to the deduced amino acid sequences of two glycosyl hydrolaseFamily 61 proteins from Trichoderma reesei (GeneSeP ADH34517) andChaetomium globosum (UniProt Q2GPR1), respectively.

An E. coli strain containing plasmid pTr3337 was deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30878, with a deposit date of Sep. 20, 2005.

Example 7 Construction of an Aspergillus oryzae Expression Vector forthe Trichoderma reesei Family GH61B Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Trichoderma reesei Family GH61B gene from the cDNA clone. AnInFusion Cloning Kit (BD Biosciences, Palo Alto, Calif.) was used toclone the fragment directly into the expression vector, pAILo2 (WO2005/074647), without the need for restriction digests and ligation.Forward primer: (SEQ ID NO: 7) 5′-ACTGGATTTACCATGAAGTCCTGCGCCATTCTTGC-3′Reverse primer: (SEQ ID NO: 8) 5′-AGTCACCTCTAGTTAGCCTTGCCACAGGGCTGG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAILo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 100 ng of Trichoderma reesei cDNA clone (prepared asdescribed in Example 5), 1× Pfx Amplification Buffer (Invitrogen,Carlsbad, Calif.), 6 μl of 10 mM blend of dATP, dTTP, dGTP, and dCTP,2.5 units of Platinum Pfx DNA Polymerase (Invitrogen, Carlsbad, Calif.),1 μl of 50 mM MgSO₄, and 5 μl of 10× pCRx Enhancer Solution (Invitrogen,Carlsbad, Calif.) in a final volume of 50 μl. The amplificationconditions were one cycle at 94° C. for 2 minutes; and 30 cycles each at94° C. for 15 seconds, 55° C. for 30 seconds, and 68° C. for 3 minutes.The heat block then went to a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using 40 mMTris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer where a 3kb product band was excised from the gel and purified using a QIAquickGel Extraction Kit (QIAGEN Inc., Valencia, Calif.) according to themanufacturer's instructions.

The fragment was then cloned into pAILo2 using an InFusion Cloning Kit.The vector was digested with Nco I and Pac I (using conditions specifiedby the manufacturer). The fragment was purified by gel electrophoresisand QIAquick gel purification. The gene fragment and the digested vectorwere combined together in a reaction resulting in the expression plasmidpTr61B (FIG. 3) in which transcription of the Family GH61B gene wasunder the control of the NA2-tpi promoter (a hybrid of the promotersfrom the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase). The recombinationreaction (20 μl) was composed of 1× InFusion Buffer (BD Biosciences,Palo Alto, Calif.), 1× BSA (BD Biosciences, Palo Alto, Calif.), 1 μl ofInFusion enzyme (diluted 1:10) (BD Biosciences, Palo Alto, Calif.), 100ng of pAILo2 digested with Nco I and Pac I, and 100 ng of theTrichoderma reesei GH61B purified PCR product. The reaction wasincubated at room temperature for 30 minutes. One μl of the reaction wasused to transform E. coli XL10 Solopac Gold cells (Stratagene, La Jolla,Calif.). An E. coli transformant containing pTr61B (GH61B gene) wasidentified by restriction enzyme digestion and plasmid DNA was preparedusing a QIAGEN BioRobot 9600.

Example 8 Expression of the Trichoderma reesei cDNA Encoding FamilyGH61B Polypeptides Having Cellulolytic Enhancing Activity in Aspergillusoryzae JaL250

Aspergillus oryzae JaL250 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Five μgof pTr61B (as well as pAILo2 as a vector control) was used to transformAspergillus oryzae JaL250.

The transformation of Aspergillus oryzae JaL250 with pTr61B (GH61B gene)yielded about 70 transformants. Ten transformants were isolated toindividual PDA plates.

Confluent PDA plates of all transformants were washed with 5 ml of 0.01%Tween 80 and inoculated separately into 25 ml of MDU2BP medium in 125 mlglass shake flasks and incubated at 34° C., 200 rpm. Five days afterincubation, 5 μl of supernatant from each culture was analyzed using8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad, Calif.)according to the manufacturer's instructions. SDS-PAGE profiles of thecultures showed that several transformants had a new major band ofapproximately 25 kDa.

A confluent plate of one transformant (grown on PDA) was washed with 10ml of 0.01% Tween 20 and inoculated into a 2 liter Fernbach containing500 ml of MDU2BP medium to generate broth for characterization of theenzyme. The flask was harvested on day 5 and filtered using a 0.22 μm GPExpress plus Membrane (Millipore, Bedford, Mass.).

Example 9 Effect of Trichoderma reesei GH61B on Hydrolysis of PretreatedCorn Stover by Trichoderma reesei Fermentation Broth ExpressingAspergillus oryzae Beta-Glucosidase

Trichoderma reesei GH61B (recombinantly produced in Aspergillus oryzaeas described in Example 8) was desalted and exchanged to 50 mM sodiumacetate pH 5.0 using a Centricon Plus-20 centrifugal filter with aBiomax-5 membrane (5000 NMWL; Millipore, Bedford, Mass.) beforehydrolysis experiments. Cell-free Trichoderma reesei fermentation brothexpressing Aspergillus oryzae beta-glucosidase, prepared as described inWO 2005/074647, was used in hydrolysis experiments without desalting orbuffer exchange.

Enzyme dilutions were prepared fresh before each experiment from stockenzyme solutions, which were stored at −20° C.

Reducing sugars (RS) were determined using a p-hydroxybenzoic acidhydrazide (PHBAH) assay (Lever, M., 1972, A new reaction forcolorimetric determination of carbohydrates, Anal. Biochem. 47:273-279), which was modified and adapted to a 96-well microplate format.

A 90 μl aliquot of the diluted sample was placed into each well of a96-well conical-bottomed microplate (Corning Inc., Acton, Mass., Costar,clear polycarbonate). The assay was started by adding 60 μl of 1.25%PHBAH in 2% sodium hydroxide to each well. The uncovered plate washeated on a custom-made heating block for 10 minutes at 95° C. After themicroplate was cooled to room temperature, 35 μl of deionized water wasadded to each well. A 100 μl aliquot was removed from each well andtransferred to a flat-bottomed 96-well plate (Corning Inc., Acton,Mass., Costar, medium binding polystyrene). The absorbance at 410 nm(A₄₁₀) was measured using a SpectraMAX Microplate Reader (MolecularDevices, Sunnyvale, Calif.). The A₄₁₀ value was translated into glucoseequivalents using a standard curve.

The standard curve was obtained with six glucose standards (0.005,0.010, 0.025, 0.050, 0.075, and 0.100 mg/ml), which were treatedsimilarly to the samples. Glucose standards were prepared by diluting 10mg/ml stock glucose solution with deionized water.

The degree of cellulose conversion to reducing sugar (conversion, %) wascalculated using the following equation: $\begin{matrix}{{Conversion}_{(\%)} = {{RS}_{({{mg}\text{/}{ml}})}*100*{162/\left( {{Cellulose}_{({{mg}\text{/}{ml}})}*180} \right)}}} \\{= {{RS}_{({{mg}\text{/}{ml}})}*{100/\left( {{Cellulose}_{({{mg}\text{/}{ml}})}*1.111} \right)}}}\end{matrix}$

In this equation, RS is the concentration of reducing sugar in solutionmeasured in glucose equivalents (mg/ml), and the factor 1.111 reflectsthe weight gain in converting cellulose to glucose.

Hydrolysis of milled PCS (1% w/v on a dry weight basis) by the cell-freeTrichoderma reesei fermentation broth expressing Aspergillus oryzaebeta-glucosidase was carried out in Deepwell Plates 96 (1.2 ml,Brinkmann, Westbury, N.Y.) capped with Deepwell Mats 96 (Brinkmann,Westbury, N.Y.). All reactions with the initial volume of 1 ml were runin 50 mM sodium acetate pH 5.0 with intermittent stirring at 50° C.

Time-course reactions containing the Trichoderma reesei fermentationbroth at 2.5 mg per g of PCS were supplemented with Trichoderma reeseiGH61B (recombinantly produced in Aspergillus oryzae as described herein)at 0.625 mg per g of PCS (25% of cellulase protein loading), and theresults were compared with non-supplemented Trichoderma reesei broth at2.5 and 3.125 mg per g of PCS or compared with Trichoderma reesei GH61Bprotein alone at 2.5 mg per g of PCS.

Protein loading reactions containing the Trichoderma reesei fermentationbroth at 2.5 mg per g of PCS were supplemented with Trichoderma reeseiGH61B protein (recombinantly expressed in Aspergillus oryzae asdescribed herein) at 0.2, 0.4, 0.6 or 1.2 mg per g PCS and incubated for115 hours. Results were compared with non-supplemented Trichodermareesei broth at 2.5 and 3.5 mg per g PCS.

Aliquots of 20 μl were removed from the PCS hydrolysis reactions atspecified time points using an 8-channel pipettor, and added to 180 μlof an alkaline mixture (102 mM Na₂CO₃ and 58 mM NaHCO₃) in MultiScreenHV 96-well filtration plate (Millipore, Bedford, Mass.) to terminate thereaction. The samples were vacuum-filtered into another flat-bottomedmicroplate to remove the PCS residue. After appropriate dilution, thefiltrates were analyzed for RS using the PHBAH assay described below

The results as shown in FIG. 4 indicated that supplementing Trichodermareesei fermentation broth expressing Aspergillus oryzae beta-glucosidasewith the Trichoderma reesei GH61B protein improved the hydrolysis yieldat all time points sampled. The improvement was greater than seen withan equal protein loading of the Trichoderma reesei broth alone. Theimprovement could not be explained by hydrolysis by the GH61Bpolypeptide alone as this amounted to only 1.2% at 115 hr even at a muchhigher protein loading of 2.5 mg per g of PCS.

The results shown in FIG. 5 indicated that the enhancement was seen overa range of protein loadings of the GH61B polypeptide, with maximalenhancement at a protein loading of approximately 16% of the proteinloading of the Trichoderma reesei broth.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession numbers:Deposit Accession Number Date of Deposit E. coli strain pTr3337 NRRLB-30878 Sep. 20, 2005

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an effective amount ofone or more cellulolytic proteins in the presence of an effective amountof a polypeptide having cellulolytic enhancing activity, wherein thepresence of the polypeptide having cellulolytic enhancing activityincreases the degradation of cellulosic material compared to the absenceof the polypeptide having cellulolytic enhancing activity and whereinthe polypeptide having cellulolytic enhancing activity is selected fromthe group consisting of: (a) a polypeptide comprising an amino acidsequence which has at least 70% identity with the mature polypeptide ofSEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide whichhybridizes under at least medium stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, or (iii) a complementary strand of (i) or (ii); (c) a polypeptidecomprising [ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ], whereinx is any amino acid, x(4,5) is any amino acid at 4 or 5 contiguouspositions, and x(3) is any amino acid at 3 contiguous positions; and (d)a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of the mature polypeptide of SEQ IDNO:
 2. 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. The method of claim1, further comprising treating the cellulosic material with an effectiveamount of one or more enzymes selected from the group consisting of ahemicellulase, esterase, protease, laccase, peroxidase, or a mixturethereof.
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. The method ofclaim 1, further comprising recovering the degraded cellulosic material.10. (canceled)
 11. (canceled)
 12. The method of claim 1, wherein thecellulolytic protein and/or the polypeptide having cellulolyticenhancing activity are in the form of a fermentation broth with orwithout cells.
 13. (canceled)
 14. (canceled)
 15. (canceled) 16.(canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled)
 20. The methodof claim 1, wherein the polypeptide having cellulolytic enhancingactivity comprises or consists of the amino acid sequence of SEQ ID NO:2 or the mature polypeptide thereof; or a fragment thereof havingcellulolytic enhancing activity.
 21. (canceled)
 22. (canceled) 23.(canceled)
 24. (canceled)
 25. The method of claim 1, wherein thepolypeptide having cellulolytic enhancing activity is encoded by thepolynucleotide contained in plasmid pTr3337 which is contained in E.coli NRRL B-30878.
 26. The method of claim 1, wherein the maturepolypeptide coding sequence of SEQ ID NO: 1 is nucleotides 77 to 766 ofSEQ ID NO:
 1. 27. The method of claims 1, wherein the mature polypeptideof SEQ ID NO: 2 is amino acids 20 to 249 of SEQ ID NO:
 2. 28. (canceled)29. A method for producing an substance, comprising: (A) saccharifying acellulosic material with an effective amount of one or more cellulolyticproteins in the presence of an effective amount of the polypeptidehaving cellulolytic enhancing activity, wherein the presence of thepolypeptide having cellulolytic enhancing activity increases thedegradation of cellulosic material compared to the absence of thepolypeptide having cellulolytic enhancing activity and wherein thepolypeptide having cellulolytic enhancing activity is selected from thegroup consisting of: (i) a polypeptide comprising an amino acid sequencewhich has at least 70% identity with the mature polypeptide of SEQ IDNO: 2; (ii) a polypeptide encoded by a polynucleotide which hybridizesunder at least medium stringency conditions with (a) the maturepolypeptide coding sequence of SEQ ID NO: 1, (b) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, or (c) a complementary strand of (a) or (b); (iii) a polypeptidecomprising [ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ], whereinx is any amino acid, x(4,5) is any amino acid at 4 or 5 contiguouspositions, and x(3) is any amino acid at 3 contiguous positions; and(iv) a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of the mature polypeptide of SEQ IDNO: 2; (B) fermenting the saccharified cellulosic material of step (a)with one or more fermentating microorganisms; and (C) recovering thesubstance from the fermentation.
 30. (canceled)
 31. (canceled) 32.(canceled)
 33. The method of claim 29, further comprising treating thecellulosic material with an effective amount of one or more enzymesselected from the group consisting of a hemicellulase, esterase,protease, laccase, peroxidase, or a mixture thereof.
 34. (canceled) 35.(canceled)
 36. The method of claim 29, wherein the substance is analcohol, organic acid, ketone, amino acid, or gas.
 37. (canceled) 38.(canceled)
 39. (canceled)
 40. (canceled)
 41. (canceled)
 42. The methodof claim 29, wherein the cellulolytic protein and/or the polypeptidehaving cellulolytic enhancing activity are in the form of a fermentationbroth with or without cells.
 43. (canceled)
 44. (canceled) 45.(canceled)
 46. (canceled)
 47. (canceled)
 48. (canceled)
 49. (canceled)50. The method of claim 29, wherein the polypeptide having cellulolyticenhancing activity comprises or consists of the amino acid sequence ofSEQ ID NO: 2 or the mature polypeptide thereof; or a fragment thereofhaving cellulolytic enhancing activity.
 51. (canceled)
 52. (canceled)53. (canceled)
 54. (canceled)
 55. The method of claim 29, wherein thepolypeptide having cellulolytic enhancing activity is encoded by thepolynucleotide contained in plasmid pTr3337 which is contained in E.coli NRRL B-30878.
 56. The method of claim 29, wherein the maturepolypeptide coding sequence of SEQ ID NO: 1 is nucleotides 77 to 766 ofSEQ ID NO:
 1. 57. The method of claims 29, wherein the maturepolypeptide of SEQ ID NO: 2 is amino acids 20 to 249 of SEQ ID NO: 2.58. (canceled)
 59. A detergent composition comprising a polypeptidehaving cellulolytic enhancing activity is selected from the groupconsisting of: (a) a polypeptide comprising an amino acid sequence whichhas at least 70% identity with the mature polypeptide of SEQ ID NO: 2;(b) a polypeptide encoded by a polynucleotide which hybridizes under atleast medium stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequencecomprising the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) a complementary strand of (i) or (ii); (c) a polypeptidecomprising [ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ], whereinx is any amino acid, x(4,5) is any amino acid at 4 or 5 contiguouspositions, and x(3) is any amino acid at 3 contiguous positions; and (d)a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of the mature polypeptide of SEQ IDNO:
 2. 60. (canceled)
 61. (canceled)
 62. (canceled)
 63. (canceled) 64.(canceled)
 65. (canceled)
 66. (canceled)
 67. The detergent compositionof claim 59, wherein the polypeptide having cellulolytic enhancingactivity comprises or consists of the amino acid sequence of SEQ ID NO:2 or the mature polypeptide thereof; or a fragment thereof havingcellulolytic enhancing activity.
 68. (canceled)
 69. (canceled) 70.(canceled)
 71. (canceled)
 72. The detergent composition of claim 59,wherein the polypeptide having cellulolytic enhancing activity isencoded by the polynucleotide contained in plasmid pTr3337 which iscontained in E. coli NRRL B-30878.
 73. The detergent composition ofclaim 59, wherein the mature polypeptide coding sequence of SEQ ID NO: 1is nucleotides 77 to 766 of SEQ ID NO:
 1. 74. The detergent compositionof claim 59, wherein the mature polypeptide of SEQ ID NO: 2 is aminoacids 20 to 249 of SEQ ID NO:
 2. 75. (canceled)